Oncofetal antigen binding proteins and related compositions and methods

ABSTRACT

The present disclosure relates to protein molecules that specifically bind to 5T4 and/or 4-1BB. The molecules may have at least one humanized 5T4-binding or 4-1BB-binding domain. Such molecules are useful for the treatment of cancer. The protein molecule binding to 5T4 or 4-1BB may have a second binding domain that binds to another target. The molecules may bind both 5T4-expressing cells and a cell-surface molecule expressed by an effector cell to enhance effector cell activation, proliferation, survival and/or effector-cell mediated cytotoxicity. The disclosure also provides pharmaceutical compositions comprising the 5T4-binding or 4-1BB-binding polypeptide or protein molecules, nucleic acid molecules encoding these polypeptides and methods of making and using these molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No. 16/041,309which claims priority to U.S. Provisional Application No. 62/535,107,filed on Jul. 20, 2017; 62/575,820, filed on Oct. 23, 2017; and62/648,072, filed on Mar. 26, 2018, the contents of which are eachincorporated herein by reference in their entireties.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety. A computer readableformat copy of the Sequence Listing (filename:APVO_057_04US_SeqList_ST25.txt, date recorded: Feb. 21, 2019, file size:260 kilobytes).

FIELD OF THE DISCLOSURE

The present disclosure relates to molecules that specifically bind totrophoblast glycoprotein (5T4). The molecules may have at least onehumanized 5T4-binding domain. These molecules are useful for thecharacterization or treatment of disorders characterized by expressionof 5T4, such as cancer. A protein therapeutic binding to 5T4 may be amonospecific protein therapeutic or a multispecific protein therapeutic.The disclosure also relates to protein therapeutics that specificallybind to tumor necrosis factor receptor superfamily member 9 (4-1BB orCD137). A multispecific protein therapeutic may bind both 5T4-expressingcells and 4-1BB expressed on effector cells to enhance effector cellactivation, proliferation, and/or effector-cell mediated cytotoxicity.

BACKGROUND OF THE DISCLOSURE

5T4 (also designated trophoblast glycoprotein, TPBG, M6P1 and Waif1) isa well-defined tumor-associated antigen (TAA) originally identified byProfessor Peter Stern, University of Manchester (Hole and Stern, 1988).It is an oncofetal antigen expressed in a high proportion of patients ina variety of malignancies, including non-small cell lung, renal,pancreas, prostate, breast, colorectal, gastric, ovarian and cervixcancers as well as in acute lymphocytic leukemia, and has also beenshown to be expressed in tumor-initiating cells (Castro et al., 2012;Damelin et al., 2011; Elkord et al., 2009; Southall et al., 1990).

Although low levels of 5T4 expression have been detected in some healthytissue, such as the placenta and specialized epithelia, expressionlevels in tumors are considerably higher.

Data suggest that 5T4 regulates the functional activity of CXCR4 (Castroet al., 2012; Southgate et al., 2010). 5T4 binding antibodies or 5T4knock-down resulted in inhibition of CXCR4-mediated cellular migration,a pathway involved in tumor growth and metastasis. Therefore, targeting5T4 may provide therapeutic benefits in the treatment of variouscancers. Currently there are no FDA approved therapeutics thatspecifically target or bind 5T4. There is a need for new therapeutics totreat malignancies in which 5T4 is expressed.

4-1BB (also known as CD137 or TNFRSF9) is a tumor necrosis factor (TNF)receptor (TNFR) superfamily member. 4-1BB is expressed on various cellpopulations including activated CD4⁺ and CD8⁺ T cells, regulatory Tcells (Treg), dendritic cells (DC), monocytes, mast cells, eosinophilsand tumor endothelial cells. Activation of 4-1BB is dependent onreceptor oligomerization (Rabu et al., 2005; Wyzgol et al., 2009)induced by binding to 4-1BBL (also known as CD137L), which is expressedas a trimer on the cell surface of antigen presenting cells (APCs) andother cell types. 4-1BB activation on CD8⁺ T cells sustains and augmentsCD8⁺ T cell effector functions and preferentially supports Th1 cytokineproduction (Shuford et al., 1997; Lee et al., 2002; Pulle et al., 2006).4-1BB activation on CD4⁺ T cells, 4-1BB stimulation initially results inactivation and later in activation-induced cell death, which is thoughtto explain why 4-1BB agonistic antibodies have shown therapeutic effectin tumor immunity as well as in autoimmunity (Zhang, J C I, 2007; Sun,Trends Mol Med, 2003). 4-1BB activation has also been reported tosuppress Treg function or convert Tregs to cytotoxic CD4⁺ T-cells(Akhmetzyanova et al., 2016; So et al., 2008).

In addition to expression on and modulation of T cell effector function,4-1BB is upregulated on CD16- and cytokine-activated natural killer (NK)cells. Activation of 4-1BB has been shown to increase antibody-dependentcellular cytotoxicity (ADCC) activity of NK cells in both murine andhuman cells (Kohrt 2012 and 2014 J Clin Invest, reviewed by Hout 2012,Oncoimm). Further, activation of 4-1BB expressed on APCs, such as DCsand macrophages may also induce and/or modulate immune activation.

The role of 4-1BB in the modulation of immune cell activation suggeststhat it may be a desirable immunotherapy target in the treatment ofmultiple cancer types. Indeed, two 4-1BB antibodies are in clinicaldevelopment: Urelumab (BMS-66513) developed by Bristol-Myers Squibb andPF-05082566 developed by Pfizer. Phase I and II studies in variousindications are ongoing for each of the antibodies. However, liver andskin toxicities have been observed in patients and murine models upon4-1BB activation (Ascierto et al., 2010; Dubrot et al., 2010; Niu etal., 2007). Further, a Phase II study with Urelumab as a second linetherapy in metastatic melanoma was terminated in 2009 due to livertoxicity (Garber et al., 2011; Li and Liu, 2013).

Therefore, there remains a need in the art for therapeutics that safelyand effectively activate 4-1BB for use in the treatment of oncologicindications.

SUMMARY OF THE DISCLOSURE

In some aspects, the disclosure relates to multispecific polypeptidesthat specifically bind to 5T4 and/or 4-1BB. In some embodiments, a5T4-binding domain of the disclosure binds to an extracellular domain ofhuman 5T4. In certain embodiments, a 4-1BB-binding domain of thedisclosure binds to an extracellular domain of human 4-1BB. In someembodiments, the disclosure provides a multispecific polypeptidecomprising a 5T4-binding domain that specifically binds to human 5T4 anda 4-1BB-binding domain, wherein said 5T4-binding domain comprises: (i)an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2,and HCDR3, and (ii) an immunoglobulin light chain variable regioncomprising LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises anamino acid sequence of SEQ ID NO: 30; (b) the HCDR2 comprises an aminoacid sequence of SEQ ID NO: 32; (c) the HCDR3 comprises an amino acidsequence of SEQ ID NO: 34; (d) the LCDR1 comprises an amino acidsequence of SEQ ID NO: 42; (e) the LCDR2 comprises an amino acidsequence of SEQ ID NO: 10; and (f) the LCDR3 comprises an amino acidsequence of SEQ ID NO: 36. In some embodiments, the 5T4-binding domaincomprising the CDR sequences recited above comprises a heavy chainvariable region comprising an amino acid sequence having at least 90%,at least 95%, or at least 97% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 38 and 46. In certain embodiments, the5T4-binding domain comprises a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 38and 46. In other embodiments, the 5T4-binding domain comprising the CDRsequences recited above comprises a light chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to a sequence selected from the group consistingof SEQ ID NOs: 44, 48, and 50. In some embodiments, the 5T4-bindingdomain comprises a light chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 44, 48, and50. In other embodiments, the 5T4-binding domain comprising the CDRsequences recited above comprises i) a heavy chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NO: 38 and a light chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NO: 44; or ii) a heavy chain variableregion comprising an amino acid sequence having at least 90%, at least95%, or at least 97% identity to SEQ ID NO: 46 and a light chainvariable region comprising an amino acid sequence having at least 90%,at least 95%, or at least 97% identity to SEQ ID NO: 48; or iii) a heavychain variable region comprising an amino acid sequence having at least90%, at least 95%, or at least 97% identity to SEQ ID NO: 46 and a lightchain variable region comprising an amino acid sequence having at least90%, at least 95%, or at least 97% identity to SEQ ID NO: 5. In someembodiments, the 5T4-binding domain comprises i) a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO: 38 and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:44; or ii) a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 46 and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 48; or iii) a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO: 46 and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:50.

In certain aspects, the disclosure includes a multispecific polypeptidecomprising a 5T4-binding domain that specifically binds to human 5T4 anda 4-1BB-binding domain that specifically binds to human 4-1BB, whereineach of the 4-1BB-binding domain and 5T4 binding domain comprise (i) animmunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2,and HCDR3; and (ii) an immunoglobulin light chain variable region (VL)comprising LCDR1, LCDR2, and LCDR3, wherein the VH and/or the VL regionof the 5T4-binding domain comprise one or more mutations in theframework region; and wherein the 5T4-binding domain comprises (a) theHCDR1 comprising an amino acid sequence of SEQ ID NO: 30; (b) the HCDR2comprising an amino acid sequence of SEQ ID NO: 32; (c) the HCDR3comprising an amino acid sequence of SEQ ID NO: 34; (d) the LCDR1comprising an amino acid sequence of SEQ ID NO: 8; (e) the LCDR2comprising an amino acid sequence of SEQ ID NO: 10; and (f) the LCDR3comprising an amino acid sequence of SEQ ID NO: 36. In some embodiments,the 5T4-binding domain comprising the CDR sequences recited abovecomprises a heavy chain variable region comprising an amino acidsequence having at least 90%, at least 95%, or at least 97% identity toSEQ ID NOs: 38. In one embodiment, the 5T4-binding domain comprises aheavy chain variable region comprising an amino acid sequence of SEQ IDNO: 38. In some embodiments, the 5T4-binding domain comprising the CDRsequences recited above comprises a light chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NO: 40. In certain embodiments, the5T4-binding domain comprises a light chain variable region comprising anamino acid sequence of SEQ ID NO: 40. In some embodiments, the5T4-binding domain comprising the CDR sequences recited above comprisesa heavy chain variable region comprising an amino acid sequence havingat least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 38and a light chain variable region comprising an amino acid sequencehaving at least 90%, at least 95%, or at least 97% identity to SEQ IDNO: 40. In some embodiments, 5T4-binding domain comprises a heavy chainvariable region comprising an amino acid sequence of SEQ ID NO: 38 and alight chain variable region comprising an amino acid sequence of SEQ IDNO: 40.

The disclosure further provides a multispecific polypeptide comprising a5T4-binding domain that specifically binds to human 5T4 and a4-1BB-binding domain that specifically binds to human 4-1BB, whereineach of the 4-1BB-binding domain and 5T4 binding domain comprise (i) animmunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2,and HCDR3; and (ii) an immunoglobulin light chain variable region (VL)comprising LCDR1, LCDR2, and LCDR3, wherein the VH and/or the VL regionof the 4-1BB-binding domain comprise one or more mutations in theframework region; and wherein the 5T4-binding domain comprises (a) theHCDR1 comprising an amino acid sequence of SEQ ID NO: 30; (b) the HCDR2comprising an amino acid sequence of SEQ ID NO: 32; c) the HCDR3comprising an amino acid sequence of SEQ ID NO: 34; (d) the LCDR1comprising an amino acid sequence of SEQ ID NO: 42; (e) the LCDR2comprising an amino acid sequence of SEQ ID NO: 10; and (f) the LCDR3comprising an amino acid sequence of SEQ ID NO: 36. In some embodiments,the 5T4-binding domain comprising the CDR sequences recited abovecomprises a heavy chain variable region comprising an amino acidsequence having at least 90%, at least 95%, or at least 97% identity toa sequence selected from the group consisting of SEQ ID NOs: 38 and 46.In certain embodiments, the 5T4-binding domain comprises a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 38 and 46. In some embodiments, the5T4-binding domain comprising the CDR sequences recited above comprisesa light chain variable region comprising an amino acid sequence havingat least 90%, at least 95%, or at least 97% identity to a sequenceselected from the group consisting of SEQ ID NOs: 44, 48, and 50. Insome aspects, the 5T4-binding domain comprises a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 44, 48, and 50. In certain embodiments, the5T4-binding domain comprising the CDR sequences recited above comprisesi) a heavy chain variable region comprising an amino acid sequencehaving at least 90%, at least 95%, or at least 97% identity to SEQ IDNO: 38 and a light chain variable region comprising an amino acidsequence having at least 90%, at least 95%, or at least 97% identity toSEQ ID NO: 44; or ii) a heavy chain variable region comprising an aminoacid sequence having at least 90%, at least 95%, or at least 97%identity to SEQ ID NO: 46 and a light chain variable region comprisingan amino acid sequence having at least 90%, at least 95%, or at least97% identity to SEQ ID NO: 48; or iii) a heavy chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NO: 46 and a light chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NO: 50. In some embodiments, the5T4-binding domain comprises i) a heavy chain variable region comprisingan amino acid sequence of SEQ ID NO: 38 and a light chain variableregion comprising an amino acid sequence of SEQ ID NO: 44; or ii) aheavy chain variable region comprising an amino acid sequence of SEQ IDNO: 46 and a light chain variable region comprising an amino acidsequence of SEQ ID NO: 48; or iii) a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO: 46 and a light chainvariable region comprising an amino acid sequence of SEQ ID NO: 50.

In some aspects, the disclosure provides a multispecific polypeptidecomprising a 5T4-binding domain that specifically binds to human 5T4 anda 4-1BB-binding domain that specifically binds to human 4-1BB, whereineach of the 4-1BB-binding domain and 5T4 binding domain comprise (i) animmunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2,and HCDR3; and (ii) an immunoglobulin light chain variable region (VL)comprising LCDR1, LCDR2, and LCDR3, wherein the VH and/or the VL regionof the 4-1BB-binding domain comprise one or more mutations in theframework region; and wherein the 5T4-binding domain comprises (a) theHCDR1 comprising an amino acid sequence of SEQ ID NO: 52; (b) the HCDR2comprising an amino acid sequence of SEQ ID NO: 32; (c) the HCDR3comprising an amino acid sequence of SEQ ID NO: 34; (d) the LCDR1comprising an amino acid sequence of SEQ ID NO: 54; (e) the LCDR2comprising an amino acid sequence of SEQ ID NO: 10; and (f) the LCDR3comprising an amino acid sequence of SEQ ID NO: 36. In certainembodiments, the 5T4-binding domain comprising the CDR sequences recitedabove comprises a heavy chain variable region comprising an amino acidsequence having at least 90%, at least 95%, or at least 97% identity toSEQ ID NO: 56. In some aspects, the 5T4-binding domain comprises a heavychain variable region comprising an amino acid sequence of SEQ ID NO:56. In certain embodiments, the 5T4-binding domain comprising the CDRsequences recited above comprises a light chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NOs: 58. In some embodiments, the5T4-binding domain comprises a light chain variable region comprising anamino acid sequence of SEQ ID NOs: 58. In certain embodiments, the5T4-binding domain comprising the CDR sequences recited above comprisesa heavy chain variable region comprising an amino acid sequence havingat least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 56and a light chain variable region comprising an amino acid sequencehaving at least 90%, at least 95%, or at least 97% identity to SEQ IDNO: 58. In some embodiments, the 5T4-binding domain comprises a heavychain variable region comprising an amino acid sequence of SEQ ID NO: 56and a light chain variable region comprising an amino acid sequence ofSEQ ID NO: 58.

The disclosure also provides a multispecific polypeptide comprising a5T4-binding domain that specifically binds to human 5T4 and a4-1BB-binding domain that specifically binds to human 4-1BB, whereineach of the 4-1BB-binding domain and 5T4 binding domain comprise (i) animmunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2,and HCDR3; and (ii) an immunoglobulin light chain variable region (VL)comprising LCDR1, LCDR2, and LCDR3, wherein the VH and/or the VL regionof the 4-1BB-binding domain comprise one or more mutations in theframework region; and wherein the 5T4-binding domain comprises (a) theHCDR1 comprising an amino acid sequence of SEQ ID NO: 60; (b) the HCDR2comprising an amino acid sequence of SEQ ID NO: 62; (c) the HCDR3comprising an amino acid sequence of SEQ ID NO: 34; (d) the LCDR1comprising an amino acid sequence of SEQ ID NO: 54; (e) the LCDR2comprising an amino acid sequence of SEQ ID NO: 10; and (f) the LCDR3comprising an amino acid sequence of SEQ ID NO: 36. In certainembodiments, the 5T4-binding domain comprising the CDR sequences recitedabove comprises a heavy chain variable region comprising an amino acidsequence having at least 90%, at least 95%, or at least 97% identity toSEQ ID NO: 64. In some embodiments, the 5T4-binding domain comprises aheavy chain variable region comprising an amino acid sequence of SEQ IDNO: 64. In certain aspects, the 5T4-binding domain comprising the CDRsequences recited above comprises a light chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NOs: 58. In some embodiments, the5T4-binding domain comprises a light chain variable region comprising anamino acid sequence of SEQ ID NOs: 58. In certain embodiments, the5T4-binding domain comprising the CDR sequences recited above comprisesa heavy chain variable region comprising an amino acid sequence havingat least 90%, at least 95%, or at least 97% identity to SEQ ID NO: 64and a light chain variable region comprising an amino acid sequencehaving at least 90%, at least 95%, or at least 97% identity to SEQ IDNO: 58. In some embodiments, the 5T4-binding domain comprises a heavychain variable region comprising an amino acid sequence of SEQ ID NO: 64and a light chain variable region comprising an amino acid sequence ofSEQ ID NO: 58.

In certain aspects, the disclosure provides a multispecific polypeptidecomprising a 4-1BB-binding domain that specifically binds to human4-1BB. The disclosure also provides a multispecific polypeptidecomprising a 4-1BB-binding domain and a 5T4-binding domain, wherein the4-1BB-binding domain is linked to the 5T4-binding domain via a bindingdomain linker. In some embodiments, a multispecific polypeptidecomprises, from amino-terminus to carboxyl-terminus, (i) a 5T4-bindingdomain, (ii) a binding domain linker, and (iii) a 4-1BB-binding domain.In certain aspects, the 5T4-binding domain is a single chain variablefragment (scFv). In some embodiments, the light chain variable region ofsaid scFv is carboxy-terminal to the heavy chain variable region of saidscFv. In other embodiments, the light chain variable region of said scFvis amino-terminal to the heavy chain variable region of said scFv. Incertain aspects, the scFv comprises a linker polypeptide. In someexamples, the linker polypeptide is between the light chain variableregion and the heavy chain variable region of said scFv. In certainembodiments, the linker polypeptide comprises a Gly₄Ser linker. In someembodiments, the linker polypeptide comprises the formula (Gly₄Ser)_(n),wherein n=1-5. In some aspects, the linker polypeptide comprises anamino acid sequence selected from SEQ ID NOs: 85-108. In certainembodiments, the linker polypeptide comprises an amino acid sequence setforth in SEQ ID NO: 98 (GGGGSGGGGSGGGGSGGGGS).

In certain embodiments, an scFv comprising the CDR sequences recitedabove comprises a sequence having at least 80%, at least 85%, at least90%, or at least 95% identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130,132, 134, and 170. In some embodiments, an scFv comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 118, 120,122, 124, 126, 128, 130, 132, 134, and 170.

In some aspects, the disclosure provides a multispecific polypeptidecomprising a 4-1BB-binding domain that specifically binds to human 4-1BBand a 5T4-binding domain, wherein said 4-1BB-binding domain comprises:(i) an immunoglobulin heavy chain variable region (VH) comprising HCDR1,HCDR2, and HCDR3, and (ii) an immunoglobulin light chain variable region(VL) comprising LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprisesan amino acid sequence of SEQ ID NO: 2; (b) the HCDR2 comprises an aminoacid sequence of SEQ ID NO: 4; (c) the HCDR3 comprises an amino acidsequence of SEQ ID NO: 6; (d) the LCDR1 comprises an amino acid sequenceof SEQ ID NO: 8; (e) the LCDR2 comprises an amino acid sequence of SEQID NO: 10; and (f) the LCDR3 comprises an amino acid sequence of SEQ IDNO: 12. In certain embodiments, the 4-1BB-binding domain comprising theCDR sequences recited above comprises a heavy chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NO: 14. In some embodiments, the4-1BB-binding domain comprises a heavy chain variable region comprisingan amino acid sequence of SEQ ID NO: 14. In certain embodiments, the4-1BB-binding domain comprising the CDR sequences recited abovecomprises a light chain variable region comprising an amino acidsequence having at least 90%, at least 95%, or at least 97% identity toSEQ ID NO: 16. In some embodiments, the 4-1BB-binding domain comprises alight chain variable region comprising an amino acid sequence of SEQ IDNO: 16. In certain embodiments, the 4-1BB-binding domain comprising theCDR sequences recited above comprises a heavy chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NO: 14 and a light chain variable regioncomprising an amino acid sequence having at least 90%, at least 95%, orat least 97% identity to SEQ ID NO: 16. In some aspects, the4-1BB-binding domain comprises a heavy chain variable region comprisingan amino acid sequence of SEQ ID NO: 14 and a light chain variableregion comprising an amino acid sequence of SEQ ID NO: 16.

The disclosure further provides a multispecific polypeptide comprising a5T4-binding domain that specifically binds to human 5T4 and a4-1BB-binding domain, wherein the 4-1BB-binding domain comprises (i) animmunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2,and HCDR3; and (ii) an immunoglobulin light chain variable region (VL)comprising LCDR1, LCDR2, and LCDR3, and wherein the VH and/or the VLregion of the 4-1BB-binding domain comprise one or more mutations in theframework region, and wherein the 4-1BB-binding domain comprises (a) theHCDR1 comprising an amino acid sequence of SEQ ID NO: 18; (b) the HCDR2comprising an amino acid sequence of SEQ ID NO: 4; (c) the HCDR3comprising an amino acid sequence of SEQ ID NO: 6; (d) the LCDR1comprising an amino acid sequence of SEQ ID NO: 8; (e) the LCDR2comprising an amino acid sequence of SEQ ID NO: 10; and (f) the LCDR3comprising an amino acid sequence of SEQ ID NO: 12. In certainembodiments, the 4-1BB-binding domain comprising the CDR sequencesrecited above comprises a heavy chain variable region comprising anamino acid sequence having at least 90%, at least 95%, or at least 97%identity to SEQ ID NO: 20. In some embodiments, the 4-1BB-binding domaincomprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 20. In certain embodiments, the 4-1BB-bindingdomain comprising the CDR sequences recited above comprises a lightchain variable region comprising an amino acid sequence having at least90%, at least 95%, or at least 97% identity to SEQ ID NO: 22. In someembodiments, the 4-1BB-binding domain comprises a light chain variableregion comprising an amino acid sequence of SEQ ID NO: 22. In certainembodiments, the 4-1BB-binding domain comprising the CDR sequencesrecited above comprises a heavy chain variable region comprising anamino acid sequence having at least 90%, at least 95%, or at least 97%identity to SEQ ID NO: 20 and a light chain variable region comprisingan amino acid sequence having at least 90%, at least 95%, or at least97% identity to SEQ ID NO: 22. In some embodiments, the 4-1BB-bindingdomain comprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 20 and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 22.

The disclosure also provides a multispecific polypeptide comprising a5T4-binding domain that specifically binds to human 5T4 and a4-1BB-binding domain, wherein the 4-1BB-binding domain comprises (i) animmunoglobulin heavy chain variable region (V_(H)) comprising HCDR1,HCDR2, and HCDR3; and (ii) an immunoglobulin light chain variable region(V_(L)) comprising LCDR1, LCDR2, and LCDR3, and wherein the V_(H) and/orthe V_(L) region of the 4-1BB-binding domain comprise one or moremutations in the framework region, and wherein the 4-1BB-binding domaincomprises (a) the HCDR1 comprising an amino acid sequence of SEQ ID NO:24; (b) the HCDR2 comprising an amino acid sequence of SEQ ID NO: 4; (c)the HCDR3 comprising an amino acid sequence of SEQ ID NO: 6; (d) theLCDR1 comprising an amino acid sequence of SEQ ID NO: 8; (e) the LCDR2comprising an amino acid sequence of SEQ ID NO: 10; and (f) the LCDR3comprising an amino acid sequence of SEQ ID NO: 12. In certainembodiments, the 4-1BB-binding domain comprising the CDR sequencesrecited above comprises a heavy chain variable region comprising anamino acid sequence having at least 90%, at least 95%, or at least 97%identity to SEQ ID NO: 26. In some embodiments, the 4-1BB-binding domaincomprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 26. In certain embodiments, the 4-1BB-bindingdomain comprising the CDR sequences recited above comprises a lightchain variable region comprising an amino acid sequence having at least90%, at least 95%, or at least 97% identity to SEQ ID NO: 16. In someembodiments, the 4-1BB-binding domain comprises a light chain variableregion comprising an amino acid sequence of SEQ ID NO: 16. In certainembodiments, the 4-1BB-binding domain comprising the CDR sequencesrecited above comprises a heavy chain variable region comprising anamino acid sequence having at least 90%, at least 95%, or at least 97%identity to SEQ ID NO: 26 and a light chain variable region comprisingan amino acid sequence having at least 90%, at least 95%, or at least97% identity to SEQ ID NO: 16. In some embodiments, the 4-1BB-bindingdomain comprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 26 and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 16.

In certain embodiments, the disclosure provides a multispecificpolypeptide comprising a 4-1BB-binding domain wherein the 4-1BB-bindingdomain is a single chain variable fragment (scFv). In some aspects, thelight chain variable region of said scFv is carboxy-terminal to theheavy chain variable region of said scFv. In other aspects, the lightchain variable region of said scFv is amino-terminal to the heavy chainvariable region of said scFv. In certain aspects, the scFv comprises alinker polypeptide. In some examples, the linker polypeptide is betweenthe light chain variable region and the heavy chain variable region ofsaid scFv. In certain embodiments, the linker polypeptide comprises aGly₄Ser linker. In some embodiments, the linker polypeptide comprisesthe formula (Gly₄Ser)_(n), wherein n=1-5. In some aspects, the linkerpolypeptide comprises an amino acid sequence selected from SEQ ID NOs:85-108. In certain embodiments, the linker polypeptide comprises anamino acid sequence set forth in SEQ ID NO: 98 (GGGGSGGGGSGGGGSGGGGS).In certain aspects, a multispecific polypeptide of the disclosurecomprises a 4-1BB-binding domain that is linked to a 5T4-binding domainvia a binding domain linker. In some embodiments, a multispecificpolypeptide comprises, from amino-terminus to carboxyl-terminus, (i) the5T4-binding domain, (ii) a binding domain linker, and (iii) the4-1BB-binding domain.

In some embodiments, the 4-1BB-binding domain comprising the CDRsequences recited above comprises a sequence having at least 80%, atleast 85%, at least 90%, or at least 95% identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 110, 112,114, and 116. In certain embodiments, the 4-1BB-binding domain comprisesa sequence selected from the group consisting of SEQ ID NOs: 110, 112,114, and 116.

In certain aspects, a multispecific polypeptide of the disclosurecomprises a 4-1BB-binding domain that is conjugated to a drug or atoxin.

The disclosure further provides a multispecific polypeptide comprising a5T4-binding domain that is fused or conjugated to an immunoglobulinconstant region. An immunoglobulin constant region may be a human Fcdomain. In some embodiments, the human Fc domain comprises a sequenceset forth in SEQ ID NO: 158 or SEQ ID NO: 160. In certain embodiments,the disclosure provides a multispecific polypeptide that comprises, fromamino-terminus to carboxyl-terminus, (i) a 5T4-binding domain, (ii) ahinge region, (iii) an immunoglobulin constant region, (iv) a bindingdomain linker, and (v) a 4-1BB-binding domain. In some embodiments, thebinding domain linker comprises a Gly₄Ser sequence. In some examples,the binding domain linker comprises the formula (Gly₄Ser)_(n), whereinn=1-5. In some aspects, the binding domain linker comprises an aminoacid sequence selected from SEQ ID NOs: 85-108. In certain embodiments,the binding domain linker comprises an amino acid sequence set forth inSEQ ID NO: 107 (SGGGGSGGGGSGGGGSPS).

In some aspects, the disclosure also provides a multispecificpolypeptide comprising a first scFv domain and a second scFv domain,wherein the first and second scFv domains are linked together by abinding domain linker or a binding domain linker and an immunoglobulinFc domain, wherein the immunoglobulin Fc domain comprises a hinge regionand an immunoglobulin constant region; and wherein the second scFvdomain specifically binds to human 4-1BB and comprises: (i) animmunoglobulin heavy chain variable region comprising an HCDR1 aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 18,24, an HCDR2 amino acid sequence of SEQ ID NO: 4, and an HCDR3 aminoacid sequence of SEQ ID NOs: 6; and (ii) an immunoglobulin light chainvariable region comprising an LCDR1 amino acid sequence of SEQ ID NOs:8, an LCDR2 amino acid sequence of SEQ ID NO: 10, and an LCDR3 aminoacid sequence of SEQ ID NO: 12, wherein the first and second scFvdomains are linked together by a binding domain linker or a bindingdomain linker and an immunoglobulin Fc domain, wherein theimmunoglobulin Fc domain comprises a hinge region and an immunoglobulinconstant region. In certain embodiments, this polypeptide comprises anamino acid sequence comprising at least 80%, at least 85%, at least 90%,or at least 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152,154, and 156. In some aspects, the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 136, 138,140, 142, 144, 146, 148, 150, 152, 154, and 156. In some embodiments,the polypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 136, 138, and 146.

In some embodiments, the multispecific polypeptide comprises, fromamino-terminus to carboxyl-terminus, (i) the first scFv domain, (ii) abinding domain linker, and (iii) the second binding domain. In otherembodiments, the multispecific polypeptide comprises, fromamino-terminus to carboxyl-terminus, (i) the second scFv domain, (ii) abinding domain linker, and (iii) the first scFv domain. In certainaspects, the multispecific polypeptide comprises, from amino-terminus tocarboxyl-terminus, (i) the first scFv domain, (ii) a hinge region, (iii)an immunoglobulin constant region, (iv) a binding domain linker, and (v)the second scFv domain. In some embodiments, the binding domain linkercomprises a Gly₄Ser sequence. In some embodiments, the binding domainlinker comprises the formula (Gly₄Ser)_(n), wherein n=1-5. In certainaspects, the binding domain linker comprises an amino acid sequenceselected from SEQ ID NOs: 85-108. In certain embodiments, the bindingdomain linker comprises an amino acid sequence set forth in SEQ ID NO:107 (SGGGGSGGGGSGGGGSPS). In some embodiments, the second scFv domain ofa multispecific polypeptide comprises: (a) the HCDR1 amino acid sequenceset forth in SEQ ID NO: 2; (b) the HCDR2 amino acid sequence set forthin SEQ ID NO: 4; (c) the HCDR3 amino acid sequence set forth in SEQ IDNO: 6; (d) the LCDR1 amino acid sequence set forth in SEQ ID NO: 8; (e)the LCDR2 amino acid sequence set forth in SEQ ID NO: 10; and (f) theLCDR3 amino acid sequence set forth in SEQ ID NO: 12. In otherembodiments, the second scFv domain of a multispecific polypeptidecomprises: (a) the HCDR1 amino acid sequence set forth in SEQ ID NO: 24;(b) the HCDR2 amino acid sequence set forth in SEQ ID NO: 4; (c) theHCDR3 amino acid sequence set forth in SEQ ID NO: 6; (d) the LCDR1 aminoacid sequence set forth in SEQ ID NO: 8; (e) the LCDR2 amino acidsequence set forth in SEQ ID NO: 10; and (f) the LCDR3 amino acidsequence set forth in SEQ ID NO: 12. In some embodiments, the secondscFv domain of a multispecific polypeptide comprises a heavy chainvariable region comprising an amino acid sequence of SEQ ID NO: 14 and alight chain variable region of SEQ ID NO: 16. In other embodiments, thesecond scFv domain of a multispecific polypeptide comprises a heavychain variable region comprising an amino acid sequence of SEQ ID NO: 20and a light chain variable region of SEQ ID NO: 22. In some embodiments,the second scFv domain of a multispecific polypeptide comprises a heavychain variable region comprising an amino acid sequence of SEQ ID NO: 26and a light chain variable region of SEQ ID NO: 16. In otherembodiments, the second scFv domain of a multispecific polypeptidecomprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 28 and a light chain variable region of SEQ IDNO: 16.

The disclosure further provides a multispecific polypeptide comprising afirst scFv domain and a second scFv domain, wherein the first and secondscFv domains are linked together by a binding domain linker or a bindingdomain linker and an immunoglobulin Fc domain, wherein theimmunoglobulin Fc domain comprises a hinge region and an immunoglobulinconstant region; wherein the second scFv domain specifically binds tohuman 4-1BB; and wherein the first scFv domain comprises (i) animmunoglobulin heavy chain variable region comprising an HCDR1 aminoacid sequence selected from the group consisting of SEQ ID NOs: 30, 52,and 60, an HCDR2 amino acid sequence selected from the group consistingof SEQ ID NOs: 32 and 62, and an HCDR3 amino acid sequence of SEQ ID NO:34; and (ii) an immunoglobulin light chain variable region comprising anLCDR1 amino acid sequence selected from the group consisting of SEQ IDNOs: 8, 42, and 54, an LCDR2 amino acid sequence of SEQ ID NOs: 10, andan LCDR3 amino acid sequence of SEQ ID NOs: 36; and wherein the secondscFv domain comprises (i) an immunoglobulin heavy chain variable regioncomprising an HCDR1 amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 18, 24, an HCDR2 amino acid sequenceselected of SEQ ID NO: 4, and an HCDR3 amino acid sequence of SEQ ID NO:6; and (ii) an immunoglobulin light chain variable region comprising anLCDR1 amino acid sequence of SEQ ID NO: 8, an LCDR2 amino acid sequenceof SEQ ID NO: 10, and an LCDR3 amino acid sequence of SEQ ID NO: 12,wherein the first and second scFv domains are linked together by abinding domain linker or a binding domain linker and an immunoglobulinFc domain, wherein the immunoglobulin Fc domain comprises a hinge regionand an immunoglobulin constant region. In some embodiments, the secondscFv domain comprises (a) the HCDR1 amino acid sequence set forth in SEQID NO: 2; (b) the HCDR2 amino acid sequence set forth in SEQ ID NO: 4;(c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 6; (d) theLCDR1 amino acid sequence set forth in SEQ ID NO: 8; (e) the LCDR2 aminoacid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3 amino acidsequence set forth in SEQ ID NO: 12. In certain embodiments, the firstscFv domain comprises: (a) the HCDR1 amino acid sequence set forth inSEQ ID NO: 30; (b) the HCDR2 amino acid sequence set forth in SEQ ID NO:32; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 34; (d)the LCDR1 amino acid sequence set forth in SEQ ID NO: 8; (e) the LCDR2amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3 aminoacid sequence set forth in SEQ ID NO: 36. In some embodiments, the firstscFv domain comprises: (a) the HCDR1 amino acid sequence set forth inSEQ ID NO: 30; (b) the HCDR2 amino acid sequence set forth in SEQ ID NO:32; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 34; (d)the LCDR1 amino acid sequence set forth in SEQ ID NO: 42; (e) the LCDR2amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3 aminoacid sequence set forth in SEQ ID NO: 36. In certain aspects, the firstscFv domain comprises: (a) the HCDR1 amino acid sequence set forth inSEQ ID NO: 30; (b) the HCDR2 amino acid sequence set forth in SEQ ID NO:32; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 34; (d)the LCDR1 amino acid sequence set forth in SEQ ID NO: 42; (e) the LCDR2amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3 aminoacid sequence set forth in SEQ ID NO: 36. In some embodiments, the firstscFv domain comprises: (a) the HCDR1 amino acid sequence set forth inSEQ ID NO: 52; (b) the HCDR2 amino acid sequence set forth in SEQ ID NO:32; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 34; (d)the LCDR1 amino acid sequence set forth in SEQ ID NO: 54; (e) the LCDR2amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3 aminoacid sequence set forth in SEQ ID NO: 36. In some embodiments, the firstscFv domain comprises: (a) the HCDR1 amino acid sequence set forth inSEQ ID NO: 60; (b) the HCDR2 amino acid sequence set forth in SEQ ID NO:62; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 34; (d)the LCDR1 amino acid sequence set forth in SEQ ID NO: 54; (e) the LCDR2amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3 aminoacid sequence set forth in SEQ ID NO: 36. In certain embodiments, thesecond scFv domain comprises: (a) the HCDR1 amino acid sequence setforth in SEQ ID NO: 18; (b) the HCDR2 amino acid sequence set forth inSEQ ID NO: 4; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO:6; (d) the LCDR1 amino acid sequence set forth in SEQ ID NO: 8; (e) theLCDR2 amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, thefirst scFv domain comprises: (a) the HCDR1 amino acid sequence set forthin SEQ ID NO: 30; (b) the HCDR2 amino acid sequence set forth in SEQ IDNO: 32; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 34;(d) the LCDR1 amino acid sequence set forth in SEQ ID NO: 42; (e) theLCDR2 amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, thesecond scFv domain comprises: (a) the HCDR1 amino acid sequence setforth in SEQ ID NO: 24; (b) the HCDR2 amino acid sequence set forth inSEQ ID NO: 4; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO:6; (d) the LCDR1 amino acid sequence set forth in SEQ ID NO: 8; (e) theLCDR2 amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, thefirst scFv domain comprises: (a) the HCDR1 amino acid sequence set forthin SEQ ID NO: 52; (b) the HCDR2 amino acid sequence set forth in SEQ IDNO: 32; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 34;(d) the LCDR1 amino acid sequence set forth in SEQ ID NO: 54; (e) theLCDR2 amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, thesecond scFv domain comprises: (a) the HCDR1 amino acid sequence setforth in SEQ ID NO: 24; (b) the HCDR2 amino acid sequence set forth inSEQ ID NO: 4; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO:6; (d) the LCDR1 amino acid sequence set forth in SEQ ID NO: 8; (e) theLCDR2 amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3amino acid sequence set forth in SEQ ID NO: 12, and, optionally, thefirst scFv domain comprises: (a) the HCDR1 amino acid sequence set forthin SEQ ID NO: 30; (b) the HCDR2 amino acid sequence set forth in SEQ IDNO: 32; (c) the HCDR3 amino acid sequence set forth in SEQ ID NO: 34;(d) the LCDR1 amino acid sequence set forth in SEQ ID NO: 8; (e) theLCDR2 amino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3amino acid sequence set forth in SEQ ID NO: 36.

The disclosure also provides a multispecific polypeptide wherein a firstscFv domain comprises i) a heavy chain variable region comprising anamino acid sequence of SEQ ID NO: 38 and a light chain variable regioncomprising an amino acid of SEQ ID NO: 40, and wherein a second scFvdomain comprises ii) a heavy chain variable region comprising an aminoacid of SEQ ID NO: 14 and a light chain variable region of SEQ ID NO:16. In some embodiments, the first scFv domain comprises i) a heavychain variable region comprising an amino acid sequence of SEQ ID NO: 38and a light chain variable region comprising an amino acid of SEQ ID NO:44, and the second scFv domain comprises ii) a heavy chain variableregion comprising an amino acid of SEQ ID NO: 14 and a light chainvariable region of SEQ ID NO: 16. In certain embodiments, the first scFvdomain comprises i) a heavy chain variable region comprising an aminoacid sequence of SEQ ID NO: 46 and a light chain variable regioncomprising an amino acid of SEQ ID NO: 48, and the second scFv domaincomprises ii) a heavy chain variable region comprising an amino acid ofSEQ ID NO: 14 and a light chain variable region of SEQ ID NO: 16. Inother embodiments, the first scFv domain comprises i) a heavy chainvariable region comprising an amino acid sequence of SEQ ID NO: 56 and alight chain variable region comprising an amino acid of SEQ ID NO: 58,and the second scFv domain comprises ii) a heavy chain variable regioncomprising an amino acid of SEQ ID NO: 14 and a light chain variableregion of SEQ ID NO: 16. In certain embodiments, the first scFv domaincomprises i) a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 64 and a light chain variable region comprisingan amino acid of SEQ ID NO: 58, and the second scFv domain comprises ii)a heavy chain variable region comprising an amino acid of SEQ ID NO: 14and a light chain variable region of SEQ ID NO: 16. In otherembodiments, the first scFv domain comprises i) a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO: 46 and a lightchain variable region comprising an amino acid of SEQ ID NO: 50, and thesecond scFv domain comprises ii) a heavy chain variable regioncomprising an amino acid of SEQ ID NO: 20 and a light chain variableregion of SEQ ID NO: 22. In yet other embodiments, the first scFv domaincomprises i) a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 56 and a light chain variable region comprisingan amino acid of SEQ ID NO: 58, and the second scFv domain comprises ii)a heavy chain variable region comprising an amino acid of SEQ ID NO: 26and a light chain variable region of SEQ ID NO: 16. In some embodiments,the first scFv domain comprises i) a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO: 38 and a light chainvariable region comprising an amino acid of SEQ ID NO: 68, and thesecond scFv domain comprises ii) a heavy chain variable regioncomprising an amino acid of SEQ ID NO: 28 and a light chain variableregion of SEQ ID NO: 16. In some embodiments, the first scFv domaincomprises i) a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 46 and a light chain variable region comprisingan amino acid of SEQ ID NO: 70, and the second scFv domain comprises ii)a heavy chain variable region comprising an amino acid of SEQ ID NO: 28and a light chain variable region of SEQ ID NO: 16.

In some embodiments, the present disclosure provides a multispecificpolypeptide comprising a first scFv domain and a second scFv domain,wherein the first and second scFv domains are linked together by abinding domain linker or a binding domain linker and an immunoglobulinFc domain, wherein the immunoglobulin Fc domain comprises a hinge regionand an immunoglobulin constant region; and wherein the first scFv domainspecifically binds to human 5T4 and comprises: (i) an immunoglobulinheavy chain variable region comprising an HCDR1 amino acid sequence ofSEQ ID NO: 30, an HCDR2 amino acid sequence of SEQ ID NO: 32, and anHCDR3 amino acid sequence of SEQ ID NO: 34; and (ii) an immunoglobulinlight chain variable region comprising an LCDR1 amino acid sequence ofSEQ ID NO: 42, an LCDR2 amino acid sequence of SEQ ID NO: 10, and anLCDR3 amino acid sequence of SEQ ID NO: 36.

In some embodiments, the first scFv domain comprises: i) a heavy chainvariable region comprising an amino acid sequence of SEQ ID NO: 38 and alight chain variable region comprising an amino acid sequence of SEQ IDNO: 44; or ii) a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 46 and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 48; or iii) a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO: 46 and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:50.

In some embodiments, the present disclosure provides a multispecificpolypeptide comprising a first scFv domain and a second scFv domain,wherein the first and second scFv domains are linked together by abinding domain linker or a binding domain linker and an immunoglobulinFc domain, wherein the immunoglobulin Fc domain comprises a hinge regionand an immunoglobulin constant region; and wherein the first scFv domainspecifically binds to human 5T4, wherein the VH and/or the VL region ofthe second scFv domain comprise one or more mutations in the frameworkregion; and wherein the first scFv domain comprises: (a) the HCDR1comprising an amino acid sequence of SEQ ID NO: 30; (b) the HCDR2comprising an amino acid sequence of SEQ ID NO: 32; (c) the HCDR3comprising an amino acid sequence of SEQ ID NO: 34; (d) the LCDR1comprising an amino acid sequence of SEQ ID NO: 42; (e) the LCDR2comprising an amino acid sequence of SEQ ID NO: 10; and (f) the LCDR3comprising an amino acid sequence of SEQ ID NO: 36. In some embodiments,the first scFv domain comprises i) a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO: 38 and a light chainvariable region comprising an amino acid sequence of SEQ ID NO: 44; orii) a heavy chain variable region comprising an amino acid sequence ofSEQ ID NO: 46 and a light chain variable region comprising an amino acidsequence of SEQ ID NO: 48; or iii) a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO: 46 and a light chainvariable region comprising an amino acid sequence of SEQ ID NO: 50.

In some embodiments, the multispecific polypeptides described hereincomprise, from amino-terminus to carboxyl-terminus, (i) the first scFvdomain, (ii) a binding domain linker, and (iii) the second bindingdomain. In some embodiments, the multispecific polypeptides comprise,from amino-terminus to carboxyl-terminus, (i) the second scFv domain,(ii) a binding domain linker, and (iii) the first scFv domain. In someembodiments, the multispecific polypeptides comprise, fromamino-terminus to carboxyl-terminus, (i) the first scFv domain, (ii) ahinge region, (iii) an immunoglobulin constant region, (iv) a bindingdomain linker, and (v) the second scFv domain. In some embodiments, thebinding domain linker comprises a Gly₄Ser sequence. In some embodiments,the binding domain linker comprises the formula (Gly₄Ser)_(n), whereinn=1-5. In some embodiments, the binding domain linker comprises an aminoacid sequence selected from SEQ ID NOs: 85-108. In some embodiments, thebinding domain linker comprises an amino acid sequence set forth in SEQID NO: 107 (SGGGGSGGGGSGGGGSPS).

In certain aspects, the first scFv domain of a multispecific polypeptideof the disclosure specifically binds to human 5T4 and comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 118,120, 122, 124, 126, 128, 130, 132, 134, and 170; and the second scFvdomain specifically binds to human 4-1BB and comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 110, 112, 114,and 116. In some embodiments, the first scFv domain comprises an aminoacid sequence set forth in SEQ ID NO: 118 and the second scFv domaincomprises an amino acid sequence set forth in SEQ ID NO: 110. In otherembodiments, the first scFv domain comprises an amino acid sequence setforth in SEQ ID NO: 120 and the second scFv domain comprises an aminoacid sequence set forth in SEQ ID NO: 110. In certain embodiments, thefirst scFv domain comprises an amino acid sequence set forth in SEQ IDNO: 122 and the second scFv domain comprises an amino acid sequence setforth in SEQ ID NO: 110. In some embodiments, the first scFv domaincomprises an amino acid sequence set forth in SEQ ID NO: 124 and thesecond scFv domain comprises an amino acid sequence set forth in SEQ IDNO: 112. In some embodiments, the first scFv domain comprises an aminoacid sequence set forth in SEQ ID NO: 126 and the second scFv domaincomprises an amino acid sequence set forth in SEQ ID NO: 114. In otherembodiments, the first scFv domain comprises an amino acid sequence setforth in SEQ ID NO: 126 and the second scFv domain comprises an aminoacid sequence set forth in SEQ ID NO: 110. In certain embodiments, thefirst scFv domain comprises an amino acid sequence set forth in SEQ IDNO: 128 and the second scFv domain comprises an amino acid sequence setforth in SEQ ID NO: 110. In some embodiments, the first scFv domaincomprises an amino acid sequence set forth in SEQ ID NO: 170 and thesecond scFv domain comprises an amino acid sequence set forth in SEQ IDNO: 116. In some embodiments, the first scFv domain comprises an aminoacid sequence set forth in SEQ ID NO: 130 and the second scFv domaincomprises an amino acid sequence set forth in SEQ ID NO: 116. In someembodiments, the first scFv domain comprises an amino acid sequence setforth in SEQ ID NO: 132 and the second scFv domain comprises an aminoacid sequence set forth in SEQ ID NO: 116. In other embodiments, thefirst scFv domain comprises an amino acid sequence set forth in SEQ IDNO: 134 and the second scFv domain comprises an amino acid sequence setforth in SEQ ID NO: 116. In some embodiments, the disclosure provides amultispecific polypeptide, wherein the polypeptide comprises an aminoacid sequence comprising at least 80%, at least 85%, at least 90%, or atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 172, 174, and 176. In some embodiments, the polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172,174, and 176.

In some embodiments, the disclosure provides a multispecific polypeptidethat specifically binds to 5T4 and 4-1BB, wherein the polypeptidecomprises an amino acid sequence comprising at least 80%, at least 85%,at least 90%, or at least 95% sequence identity to a sequence selectedfrom the group consisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 172, 174, and 176. In certain embodiments, thepolypeptide that specifically binds to 5T4 and 4-1BB comprises an aminoacid sequence comprising at least 80%, at least 85%, at least 90%, or atleast 95% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 172, 174, and 176, wherein the polypeptide comprises the sameCDR amino acid sequences as the respective SEQ ID NO or the polypeptidecomprises CDR amino acid sequences that deviate by no more than oneamino acid as compared to the respective SEQ ID NO. In some embodiments,the polypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 172, 174, and 176.

The disclosure further provides a multispecific polypeptide wherein thefirst scFv domain binds to an extracellular domain of 5T4 and whereinthe second scFv domain binds to an extracellular domain of 4-1BB. Insome aspects, the polypeptide results in enhanced effector cellactivation. In some aspects, a multispecific polypeptide of thedisclosure increases effector cell activation and/or effector cellproliferation. In other aspects, a multispecific polypeptide enhanceseffector cell-dependent lysis of 5T4-expressing cells. In someembodiments, a 5T4-binding domain of the disclosure is capable ofbinding 5T4 with a kD value of less than 50 nM. In some aspects, thelight chain variable region and/or the heavy chain variable region ofthe 5T4-binding domain and/or the 4-1BB-binding domain is humanized.

In some embodiments, the present disclosure provides a multispecificpolypeptide comprising a first single chain variable fragment (scFv)domain and a second scFv domain linked together by a binding domainlinker and an immunoglobulin Fc domain, wherein the immunoglobulin Fcdomain comprises a hinge region and an immunoglobulin constant region;wherein the multispecific polypeptide is capable of forming a homodimerby association with a second identical multispecific polypeptide;wherein the first scFv domain specifically binds to human 5T4 andcomprises: (i) an immunoglobulin heavy chain variable region comprisinga heavy chain complementarity determining region (HCDR)-1 amino acidsequence of SEQ ID NO: 30, an HCDR2 amino acid sequence of SEQ ID NO:32, and an HCDR3 amino acid sequence of SEQ ID NO: 34; and (ii) animmunoglobulin light chain variable region comprising a light chaincomplementarity determining region (LCDR)-1 amino acid sequence of SEQID NO: 42, an LCDR2 amino acid sequence of SEQ ID NO: 10, and an LCDR3amino acid sequence of SEQ ID NOs: 36; and wherein the second scFvdomain specifically binds to human 4-1BB and comprises: (i) animmunoglobulin heavy chain variable region comprising an HCDR1 aminoacid sequence of SEQ ID NO: 2, an HCDR2 amino acid sequence of SEQ IDNO: 4, and an HCDR3 amino acid sequence of SEQ ID NO: 6; and (ii) animmunoglobulin light chain variable region comprising an LCDR1 aminoacid sequence of SEQ ID NO: 8, an LCDR2 amino acid sequence of SEQ IDNO: 10, and an LCDR3 amino acid sequence of SEQ ID NO: 12. 2. Themultispecific polypeptide of claim 1, wherein the multispecificpolypeptide comprises, from amino-terminus to carboxyl-terminus: (i) thefirst scFv domain, (ii) the hinge region, (iii) the immunoglobulinconstant region, (iv) the binding domain linker, and (v) the second scFvdomain. In some embodiments, the first scFv domain comprises a mutationin the framework region compared to the framework region of SEQ ID NOs:130 or 170. In some embodiments, the mutation introduces a stabilizingdisulfide bond.

In some embodiments, the first scFv domain of the multispecificpolypeptide comprises the immunoglobulin heavy chain variable region ofSEQ ID NO: 38 and the immunoglobulin light chain variable region of SEQID NO: 44. In some embodiments, the first scFv domain of themultispecific polypeptide comprises the immunoglobulin heavy chainvariable region of SEQ ID NO: 46 and the immunoglobulin light chainvariable region of SEQ ID NO:48. In some embodiments, the second scFvdomain of the multispecific polypeptide comprises immunoglobulin heavychain variable region of SEQ ID NO: 14 and the immunoglobulin lightchain variable region of SEQ ID NO: 16. In some embodiments, the firstscFv domain of the multispecific polypeptide comprises theimmunoglobulin heavy chain variable region of SEQ ID NO: 46 and theimmunoglobulin light chain variable region of SEQ ID NO: 48, and thesecond scFv domain comprises the immunoglobulin heavy chain variableregion of SEQ ID NO: 14 and the immunoglobulin light chain variableregion of SEQ ID NO: 16. In some embodiments, the amino acid sequence ofthe first scFv domain is at least 97% identical to SEQ ID NO: 120, andthe amino acid sequence of the second scFv domain is at least 97%identical to SEQ ID NO: 110. In some embodiments, the polypeptidecomprises an amino acid sequence at least 95% identical to SEQ ID NO:172 or comprises an amino acid sequence identical to SEQ ID NO: 172. Insome embodiments, the amino acid sequence of the first scFv domain is atleast 97% identical to SEQ ID NO: 122 and the amino acid sequence of thesecond scFv domain is at least 97% identical to SEQ ID NO: 110. In someembodiments, the polypeptide comprises an amino acid sequence at least95% identical to SEQ ID NO: 174 or comprises an amino acid sequence ofSEQ ID NO: 174.

In some embodiments, binding of the multispecific polypeptide to aneffector cell results in increased effector cell activation, increasedeffector cell proliferation, or wherein binding of the multispecificpolypeptide to an effector cell an a 5T4-expressing cell results inenhanced effector cell-dependent lysis of the 5T4-expressing cell.

The disclosure also encompasses a dimer comprising two identicalpolypeptides, wherein the two polypeptides are each the multispecificpolypeptide described in the disclosure.

In certain aspects, the disclosure provides a pharmaceutical compositioncomprising a polypeptide or a protein described in the disclosure, and apharmaceutically acceptable carrier, diluent, or excipient. In someembodiments, a pharmaceutical composition may be formulated in a dosageform selected from the group consisting of: an oral unit dosage form, anintravenous unit dosage form, an intranasal unit dosage form, asuppository unit dosage form, an intradermal unit dosage form, anintramuscular unit dosage form, an intraperitoneal unit dosage form, asubcutaneous unit dosage form, an epidural unit dosage form, asublingual unit dosage form, and an intracerebral unit dosage form.Non-limiting examples of an oral unit dosage form include tablets,pills, pellets, capsules, powders, lozenges, granules, solutions,suspensions, emulsions, syrups, elixirs, sustained-release formulations,aerosols, and sprays.

The disclosure further encompasses a method for enhancing effector cellactivation against a cell expressing 5T4, the method comprising:contacting said 5T4-expressing cell with a polypeptide or a protein ofthe disclosure, wherein said contacting is under conditions wherebyenhanced effector cell activation against the 5T4-expressing cell isinduced. In some aspects, the disclosure provides a method for treatinga disorder in a subject, wherein said disorder is characterized byexpression of 5T4, the method comprising administering to the subject atherapeutically effective amount of a polypeptide or a protein or apharmaceutical composition of the disclosure. The disclosure alsoencompasses a use of a polypeptide or a protein of the disclosure forthe manufacture of a medicament for treatment of a disorder in asubject, wherein said disorder is characterized by expression of 5T4. Insome embodiments, the disclosure is related to a polypeptide or aprotein of the disclosure for use in treating a disorder in a subject,wherein said disorder is characterized by expression of 5T4. In someembodiments, the disorder is a cancer. In certain aspects, the cancer isbreast cancer, pancreatic cancer, ovarian cancer, non-small cell lungcancer, mesothelioma, chronic lymphocytic leukemia (CLL), mantle cellleukemia (MCL), acute lymphoblastic leukemia (ALL), squamous cellcarcinoma, melanoma, adrenal cancer, bladder cancer, cervical cancer,renal cancer, gastric cancer, prostate cancer, thyroid cancer, livercancer, uterine cancer, neurofibroma, sarcoma or head and neck cancer.

In some embodiments, the multispecific polypeptide for use in suchmethods comprises, from amino-terminus to carboxyl-terminus: (i) thefirst scFv domain, (ii) the hinge region, (iii) the immunoglobulinconstant region, (iv) the binding domain linker, and (v) the second scFvdomain. In some embodiments, the multispecific polypeptide for use insuch methods comprisines a second scFv domain comprising animmunoglobulin heavy chain variable region of SEQ ID NO: 14 and animmunoglobulin light chain variable region of SEQ ID NO: 16, and a firstscFv domain comprising (i) the immunoglobulin heavy chain variableregion of SEQ ID NO: 38 and the immunoglobulin light chain variableregion of SEQ ID NO: 44; or (ii) the immunoglobulin heavy chain variableregion of SEQ ID NO: 46 and the immunoglobulin light chain variableregion of SEQ ID NO: 48. In some embodiments, the amino acid sequence ofthe second scFv domain of the multispecific polypeptide is at least 97%identical to SEQ ID NO: 110, and the amino acid sequence of the firstscFv domain of the multispecific polypeptide is at least 97% identicalto SEQ ID NO: 120 (anti-5T4 scFv for 209) or SEQ ID NO: 122. In someembodiments, said polypeptide exhibits statistically significantenhanced effector cell activation compared to a second multispecificpolypeptide, wherein the second multispecific polypeptide is an IgG-scFvstructure comprising an anti-4-1BB antibody comprising a variable heavychain comprising SEQ ID NO: 28 and a variable light chain comprising SEQID NO: 16 and an anti-5T4 scFv comprising a variable heavy chaincomprising SEQ ID NO: 46 and a variable light chain comprising SEQ IDNO: 66. In some embodiments, said polypeptide induces statisticallysignificant increased effector cell proliferation compared to a secondmultispecific polypeptide, wherein the second multispecific polypeptideis an IgG-scFv structure comprising an anti-4-1BB antibody comprising avariable heavy chain comprising SEQ ID NO: 28 and a variable light chaincomprising SEQ ID NO: 16 and an anti-5T4 scFv comprising a variableheavy chain comprising SEQ ID NO: 46 and a variable light chaincomprising SEQ ID NO: 66.

Some aspects of the disclosure include an isolated nucleic acid moleculeencoding a polypeptide of the disclosure. In some embodiments, a nucleicacid molecule comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 171, 173, and 175. The disclosure encompasses an expression vectorcomprising an isolated nucleic acid molecule described herein. In someembodiments, the nucleic acid molecule in an expression vector isoperatively linked to regulatory sequences suitable for expression ofthe nucleic acid segment in a host cell. The disclosure provides arecombinant host cell comprising an expression vector described herein.In some aspects, the disclosure provides a method for producing apolypeptide comprising a 5T4-binding domain, the method comprisingculturing a recombinant host cell comprising an expression vectordescribed herein under conditions whereby the nucleic acid segment isexpressed, thereby producing the polypeptide comprising a 5T4-bindingdomain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the agonistic function of seven constructs(ALG.APV-004, ALG.APV-006, ALG.APV-099, ALG.APV-127, ALG.APV-148, andALG.APV-150) in the presence of 5T4(+) cells.

FIG. 2 illustrates binding curves of seven scFv-Fc-scFv molecules(ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191,ALG.APV-196, and ALG.APV-198) to the CHO-K1 cell line expressing human4-1BB.

FIG. 3 illustrates binding curves of seven scFv-Fc-scFv molecules(ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191,ALG.APV-196 and ALG.APV-198) to the CHO-K1 cell line expressingcynomolgus 4-1BB.

FIG. 4 illustrates the binding curves of seven scFv-Fc-scFv molecules(ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191,ALG.APV-196, and ALG.APV-198) to the CHO-K1 cell line expressing human5T4.

FIG. 5 illustrates the expression levels of human 5T4 in the CHO-K1 andSKOV-3 cell lines.

FIG. 6 illustrates the binding curves of the ALG.APV-006, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-196, and ALG.APV-198 constructs onSKOV-3 cells.

FIG. 7 illustrates the binding curves of ALG.APV-006, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196, and ALG.APV-198 onthe CHO-K1 cell line expressing cynomolgus 5T4.

FIG. 8 illustrates the activity of anti-5T4×anti-4-1BB constructs in a4-1BB reporter assay.

FIG. 9 illustrates the effect of anti-5T4×anti-4-1BB constructs on IFNγrelease from PBMC and CHO-K1 co-cultures.

FIG. 10 illustrates an exemplary embodiment of a protein with anscFv-Fc-scFv format (ADAPTIR™ format).

FIG. 11A-FIG. 11B show the interferon gamma (IFNγ) response in humanCD8+ T cells cultured with bispecific scFv-Fc-scFv constructs. FIG. 11Ashows T cell responses when cultured in plates coated with 5T4-Fcantigen. FIG. 11B shows T cell responses when cultured without 5T4-Fcantigen. The figure displays mean values from two donors.

FIG. 12 shows the binding curves of ALG.APV-004, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209, and ALG.APV-210 tothe CHO-K1/human CD137 cell line.

FIG. 13 shows the binding curves of ALG.APV-004, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209, and ALG.APV-210 tomurine CT26 cells (ATCC) expressing human 5T4.

FIG. 14 shows the binding curves of ALG.APV-004, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 onthe CHO-K1/cynomolgus 5T4 transfectants.

FIG. 15 shows the binding curves of ALG.APV-004, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 on ahuman cell line expressing neither CD137 nor 5T4 (MOLM13 cells, ATCC).

FIG. 16A-FIG. 16B show the activity of the bispecific constructs inJurkat/NF-κB reporter cells, after 5 hours of incubation in the presenceof 5T4(+) cells (HCC1143, FIG. 16A) or 5T4(−) cells (MOLM13, FIG. 16B).Every point in the curve represents the average of duplicate wells. They-axis shows values in relative fluorescence units (RLU).

FIG. 17 shows the levels of IFN-γ induced in primary PBMC cultures at 72by ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209,ALG.APV-210, and the Morrison format control construct, ALG.APV-004.Every point in the curve represents the average of duplicate wells.

FIG. 18A-FIG. 18B illustrates FACS analysis of 5T4-dependentlocalization of bispecific constructs ALG.APV-209, ALG.APV-210, andALG.APV-004 to antigen-expressing tumors. Localization was detected bystaining with an anti-human Fc antibody (FIG. 18A) or biotinylated 4-1BB(FIG. 18B).

FIG. 19A-FIG. 19B illustrates immunohistochemical analysis of5T4-dependent localization of bispecific constructs ALG.APV-209,ALG.APV-210, and ALG.APV-004 to 5T4+(FIG. 19A) and 5T4-(FIG. 19B)tumors.

FIG. 20 shows the IFNγ response of human CD8 T cells cultured with thebispecific antibody ALG.APV-210 in the presence of CT26 cells expressingdifferent levels of 5T4.

FIG. 21A-FIG. 21B show the maximum IFNγ release of human CD8 T cellscultured with the bispecific antibody ALG.APV-210 in the presence ofCT26 cells expressing different levels of 5T4.

FIG. 22A-FIG. 22C show ALG.APV-210 or ANC107 (isotype control) bindingto primary CD8 T cells gated from CD3-stimulated or unstimulated humanand cynomolgus PBMC. The MFI (and SEM) from 2 independent experimentsare shown in FIG. 22A and FIG. 22B. FIG. 22C shows the normalized pooleddata for ALG.APV-210 from the 2 independent experiments. n=3donors/group/exp.

FIG. 23A-FIG. 23C show ALG.APV-210 or ANC107 (isotype control) bindingto primary CD8 T cells gated from CD3-stimulated or unstimulated humanand cynomolgus PBMC. Percentage and SEM of ALG.APV-210 binding to CD8 Tcells from 2 independent experiments are shown in FIGS. 23A and 23B.FIG. 23C shows the normalized pooled data from experiment 1 and 2. n=3donors/group/exp.

FIG. 24 shows the agonistic function of ALG.APV-210 on human CD8 Tcells.

FIG. 25A-FIG. 25B show the agonistic function of ALG.APV-210 on humanCD8 T cells from individual representative donors (FIG. 25A and FIG.25B).

FIG. 26 shows the agonistic function of ALG.APV-210 on cynomolgus CD8 Tcells.

FIG. 27A-FIG. 27C show the agonistic function of ALG.APV-210 oncynomolgus CD8 T cells from individual representative donors (FIG. 27A,FIG. 27B, and FIG. 27C). The figures show a dose response dependent IFNγproduction by cynomolgus CD8 T activated with ALG.APV-210 in thepresence of 5T4-Fc.

FIG. 28 shows the anti-tumor efficacy of a bispecific antibodyALG.APV-210 in HCT-116, a 5T4 positive human colon carcinoma xenografttumor model in SCID beige mice.

FIG. 29 shows the PK analysis of ALG.APV-210 or ALG.APV-209.

FIG. 30A-FIG. 30B show the binding of the Fc portion of ALG.APV-210 toFcγR. Titrated ALG.APV-210 was incubated with the FcγR-expressing cellsand probed with a fluorescently-labelled anti-human IgG secondaryantibody (FIG. 30A). Human IgG1 molecule was used as a positive control(FIG. 30B).

FIG. 31A-FIG. 31B show the CD8+ (FIG. 31A) and CD4+ (FIG. 31B) T-cellproliferation induced by the bispecific molecules ALG.APV-209 andALG.APV-210 in the presence of CHO-K1 cells expressing human 5T4 andanti-CD3 antibodies.

FIG. 32A-FIG. 32B show induction of IFN-γ secretion by the bispecificmolecule ALG.APV-210 in whole PBMC cultures, from two different humanPBMC donors (FIG. 32A and FIG. 32B).

FIG. 33A-FIG. 33B show CD8+ T-cell proliferation induced by thebispecific molecule ALG.APV-210 in whole PBMC cultures, from twodifferent human PBMC donors (FIG. 33A and FIG. 33B).

FIG. 34 shows binding of ALG.APV-210 to 5T4-expressing cell lines.

FIG. 35 shows agonist function of the bispecific construct ALG.APV-210in Jurkat/NF-κB reporter cells after incubation in the presence of cellsexpressing a range of surface 5T4 protein densities.

FIG. 36 shows the IFNγ response of human CD8+ T cells cultured with thebispecific antibody ALG.APV-210 in the presence of human HCT116 cellsexpressing 5T4.

FIG. 37 shows IFNγ response of human CD8+ T cells cultured with thebispecific antibody ALG.APV-210 in the presence of human HCT116 cellsexpressing 5T4.

FIG. 38 shows the maximum IFNγ release of human CD8 T cells culturedwith the bispecific antibody ALG.APV-210 in the presence of HCT116 cellsexpressing human 5T4.

FIG. 39A-FIG. 39D show ALG.APV-210 binding to 5T4 expressing human tumorcells (FIG. 39A and FIG. 39B) or transfected cell lines (FIG. 39C andFIG. 39D).

FIG. 40A-FIG. 40D show normalized MFI for experiments demonstratingALG.APV-210 binding to 5T4 expressing human tumor cells (FIG. 40A andFIG. 40B) or transfected cell lines (FIG. 40C and FIG. 40D).

DETAILED DESCRIPTION OF THE DISCLOSURE

The disclosure provides polypeptides comprising binding domains thatspecifically bind to trophoblast glycoprotein (5T4) and polypeptidesthat specifically bind to 5T4. In some embodiments, the polypeptides aremulti specific polypeptides that may bind specifically to 5T4 and toanother target. In some embodiments, the polypeptides described hereinare multispecific polypeptides that bind specifically to 5T4 and alsobind specifically to a target on an effector cell. The disclosure alsoprovides polypeptides comprising binding domains that specifically bindto tumor necrosis factor receptor superfamily member 9 (41BB or CD137)and polypeptides that specifically bind to 4-1BB. In some embodiments,the polypeptides are multispecific polypeptides that may bindspecifically to 4-1BB and to another target (e.g., a tumor-associatedantigen). In some embodiments, the polypeptides described herein aremultispecific polypeptides that bind specifically to 4-1BB and also bindspecifically to a target on a target cell. In some embodiments, themultispecific polypeptides are bispecific polypeptides that bindspecifically to 5T4 and bind specifically to 4-1BB. In some embodiments,the bispecific polypeptides bind to 5T4 expressed on a target cell and4-1BB expressed on an effector cell, thereby resulting in amplificationof effector cell activation and enhancing effector cell-mediatedcytotoxicity of a target cell.

In some embodiments, the present disclosure provides 5T4-binding domainsand/or 4-1BB-binding domains (and polypeptides or proteins comprisingsuch binding domains) that exhibit less off-target binding relative topreviously known 5T4-binding domains and/or 4-1BB-binding domains. Incertain aspects, the binding domains and/or polypeptides comprising thebinding domains described herein bind to 5T4 and/or 4-1BB moreeffectively in certain formats and/or certain orientations (e.g.,V_(H)-V_(L) compared to V_(L)-V_(H)), leading to higher potency and/orimproved utility in treating disorders associated with expression of5T4.

In some embodiments, administration of a therapeutically effectiveamount of a polypeptide or protein described herein to a patient in needthereof is useful for treatment of certain disorders associated with theexpression of 5T4, including certain cancers. In one embodiment, thepolypeptide or protein binds both a target cell expressing 5T4 and aneffector-cell, thereby “cross-linking” the target cell expressing 5T4and the effector cell. The binding of both domains to their targetsenhances the activation of the effector cells, leading to a prolongedand/or more robust effector cell response (e.g., effector cell-mediatedcytotoxicity). The polypeptides and proteins of the present disclosureoffer various advantages in treating patients, for example, effectivebinding to 5T4, efficient enhancement of effector cell activity, reducedlevels of cytokine release, and/or a lower risk of adverse events (e.g.,toxicity). In some embodiments, a target cell expresses 5T4 at a higherlevel than a non-target cell (e.g., normal cell or non-cancerous cell inthe same subject, organ, or tissue) expresses 5T4. In other embodiments,a target cell expresses 5T4 while a non-target cell (e.g., normal cellor non-cancerous cell in the same subject, organ, or tissue) does notexpress 5T4.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited herein, including but notlimited to patents, patent applications, articles, books, and treatises,are hereby expressly incorporated by reference in their entirety for anypurpose. In the event that one or more of the incorporated documents orportions of documents define a term that contradicts that term'sdefinition in the application, the definition that appears in thisapplication controls. However, mention of any reference, article,publication, patent, patent publication, and patent application citedherein is not, and should not be taken as an acknowledgment, or any formof suggestion, that they constitute valid prior art or form part of thecommon general knowledge in any country in the world.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. It should be understood that the terms “a” and “an”as used herein refer to “one or more” of the enumerated componentsunless otherwise indicated. The use of the alternative (e.g., “or”)should be understood to mean either one, both, or any combinationthereof of the alternatives. As used herein, the terms “include” and“comprise” are used synonymously. In addition, it should be understoodthat the polypeptides comprising the various combinations of thecomponents (e.g., domains or regions) and substituents described herein,are disclosed by the present application to the same extent as if eachpolypeptide was set forth individually. Thus, selection of particularcomponents of individual polypeptides is within the scope of the presentdisclosure.

Definitions

As used herein, the term “binding domain” or “binding region” refers tothe domain, region, portion, or site of a protein, polypeptide,oligopeptide, peptide, antibody, or binding domain derived from anantibody that possesses the ability to specifically recognize and bindto a target molecule, such as an antigen, ligand, receptor, substrate,or inhibitor (e.g., 5T4 or 4-1BB). Exemplary binding domains include,antibodies and antibody-like proteins or domains, antibody heavy andlight chain variable regions, and single-chain antibody variable regions(e.g., domain antibodies, sFv, scFv, scFab). In certain embodiments, thebinding domain comprises or consists of an antigen binding site (e.g.,comprising a variable heavy chain sequence and variable light chainsequence or three light chain complementary determining regions (CDRs)and three heavy chain CDRs from an antibody placed into alternativeframework regions (FRs) (e.g., human FRs optionally comprising one ormore amino acid substitutions). A variety of assays are known foridentifying binding domains of the present disclosure that specificallybind a particular target, including Western blot, ELISA, phage displaylibrary screening, and BIACORE® interaction analysis. In someembodiments, the polypeptides of the present invention comprise abinding domain that specifically binds to a target antigen expressed bya target cell (e.g., a tumor associated antigen, such as 5T4). In someembodiments, the polypeptides of the present invention comprise abinding domain that specifically binds to a target antigen expressed byan effector cell (e.g., 4-1BB). In some embodiments, the polypeptides ofthe present invention are multispecific polypeptides and comprise two ormore binding domains.

A binding domain or protein comprising a binding domain “specificallybinds” a target if it binds the target with an affinity or K_(a) (i.e.,an equilibrium association constant of a particular binding interactionwith units of 1/M) equal to or greater than 10⁵ M⁻¹, while notsignificantly binding other components present in a test sample. Bindingdomains can be classified as “high affinity” binding domains and “lowaffinity” binding domains. “High affinity” binding domains refer tothose binding domains with a K_(a) of at least 10⁷ M⁻¹, at least 10⁸M⁻¹, at least 10⁹ M⁻¹, at least 10¹⁰ M⁻, at least 10¹¹ M⁻¹, at least10¹² M⁻¹, or at least 10¹³ M⁻¹. “Low affinity” binding domains refer tothose binding domains with a K_(a) of up to 10⁷ M⁻¹, up to 10⁶ M⁻¹, upto 10⁵ M⁻¹. Alternatively, affinity can be defined as an equilibriumdissociation constant (K_(d)) of a particular binding interaction withunits of M (e.g., 10⁻⁵ M to 10⁻¹³, or about 500 nM, about 300 nM, about250 nM, about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 25nM, about 10 nM, or about 5 nM). Affinities of binding domainpolypeptides and single chain polypeptides according to the presentdisclosure can be readily determined using conventional techniques (see,e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660; and U.S. Pat.Nos. 5,283,173, 5,468,614, or the equivalent).

As used herein, a “conservative substitution” is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are well-knownin the art (see, e.g., PCT Application Publication No. WO 97/09433, page10, published Mar. 13, 1997; Lehninger, Biochemistry, Second Edition;Worth Publishers, Inc. NY:NY (1975), pp. 71-77; Lewin, Genes IV, OxfordUniversity Press, NY and Cell Press, Cambridge, Mass. (1990), p. 8).

As used herein, the term “derivative” refers to a modification of one ormore amino acid residues of a peptide by chemical or biological means,either with or without an enzyme, e.g., by glycosylation, alkylation,acylation, ester formation, or amide formation.

As used herein, a polypeptide or amino acid sequence “derived from” adesignated polypeptide or protein refers to the origin of thepolypeptide. In certain embodiments, the polypeptide or amino acidsequence which is derived from a particular sequence (sometimes referredto as the “parent” or “parental” sequence) and has an amino acidsequence that is essentially identical to the parent sequence or aportion thereof, wherein the portion consists of at least 10-20 aminoacids, at least 20-30 amino acids, or at least 30-50 amino acids, or atleast 50-150 amino acids, or which is otherwise identifiable to one ofordinary skill in the art as having its origin in the parent sequence.For example, a binding domain (e.g., a Fab, F(ab′)2, Fab′, scFv, singledomain antibody (sdAb), etc.) can be derived from an antibody. In someembodiments, a binding domain sequence (e.g., a 5T4- or 4-1BB-bindingdomain) is derived from an antibody or protein by means of a computeralgorithm or in silico.

Polypeptides derived from another polypeptide can have one or moremutations or alterations relative to the parent polypeptide, e.g., oneor more amino acid residues which have been substituted with anotheramino acid residue or which has one or more amino acid insertions ordeletions. In such embodiments, polypeptides derived from a parentpolypeptide and comprising one or more mutations or alteration arereferred to as “variants.” As used herein, the term “variant” or“variants” refers to a polynucleotide or polypeptide with a sequencediffering from that of a reference polynucleotide or polypeptide, butretaining essential properties thereof. Generally, variantpolynucleotide or polypeptide sequences are overall closely similar,and, in many regions, identical to the reference polynucleotide orpolypeptide. For instance, a variant polynucleotide or polypeptide mayexhibit at least about 70%, at least about 80%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98% or at least about 99% sequence identity compared to theactive portion or full length reference polynucleotide or polypeptide.The polypeptide can comprise an amino acid sequence which is notnaturally occurring. Such variations necessarily have less than 100%sequence identity or similarity with the parent polypeptide. In oneembodiment, the variant will have an amino acid sequence from about 60%to less than 100% amino acid sequence identity or similarity with theamino acid sequence of the parent polypeptide. In another embodiment,the variant will have an amino acid sequence from about 75% to less than100%, from about 80% to less than 100%, from about 85% to less than100%, from about 90% to less than 100%, from about 95% to less than 100%amino acid sequence identity or similarity with the amino acid sequenceof the parent polypeptide.

As used herein, the term “sequence identity” refers to a relationshipbetween two or more polynucleotide sequences or between two or morepolypeptide sequences. When a position in one sequence is occupied bythe same nucleic acid base or amino acid residue in the correspondingposition of the comparator sequence, the sequences are said to be“identical” at that position. The percentage sequence identity iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of identical positions. The number of identicalpositions is then divided by the total number of positions in thecomparison window and multiplied by 100 to yield the percentage ofsequence identity. Percentage of sequence identity is determined bycomparing two optimally aligned sequences over a comparison window. Thecomparison window for polynucleotide sequences can be, for instance, atleast 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or morenucleic acids in length. The comparison window for polypeptide sequencescan be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids inlength. In order to optimally align sequences for comparison, theportion of a polynucleotide or polypeptide sequence in the comparisonwindow can comprise additions or deletions termed gaps while thereference sequence is kept constant. An optimal alignment is thatalignment which, even with gaps, produces the greatest possible numberof “identical” positions between the reference and comparator sequences.Percentage “sequence identity” between two sequences can be determinedusing the version of the program “BLAST 2 Sequences” which was availablefrom the National Center for Biotechnology Information as of Sep. 1,2004, which program incorporates the programs BLASTN (for nucleotidesequence comparison) and BLASTP (for polypeptide sequence comparison),which programs are based on the algorithm of Karlin and Altschul (Proc.Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing “BLAST 2Sequences,” parameters that were default parameters as of Sep. 1, 2004,can be used for word size (3), open gap penalty (11), extension gappenalty (1), gap dropoff (50), expect value (10) and any other requiredparameter including but not limited to matrix option. Two nucleotide oramino acid sequences are considered to have “substantially similarsequence identity” or “substantial sequence identity” if the twosequences have at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity relative to each other.

As used herein, unless otherwise provided, a position of an amino acidresidue in a variable region of an immunoglobulin molecule is numberedaccording to either the IMGT criteria (Brochet et al, Nucl. Acids Res.(2008) 36, W503-508) or according to EU nomenclature (Ward et al., 1995Therap. Immunol. 2:77-94), and a position of an amino acid residue in aconstant region of an immunoglobulin molecule is numbered according toEU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94). The Kabatnumbering convention (Kabat, Sequences of Proteins of ImmunologicalInterest, 5^(th) ed. Bethesda, Md.: Public Health Service, NationalInstitutes of Health (1991)) is an alternative system used to refer to aposition of an amino acid residue in a variable region of animmunoglobulin molecule and is sometimes used to refer to a position ofan amino acid residue in a variable region of an immunoglobulin moleculeherein.

As used herein, the term “dimer” refers to a biological entity thatconsists of two subunits associated with each other via one or moreforms of intramolecular forces, including covalent bonds (e.g.,disulfide bonds) and other interactions (e.g., electrostaticinteractions, salt bridges, hydrogen bonding, and hydrophobicinteractions), and is stable under appropriate conditions (e.g., underphysiological conditions, in an aqueous solution suitable forexpressing, purifying, and/or storing recombinant proteins, or underconditions for non-denaturing and/or non-reducing electrophoresis). Theterms “heterodimer” or “heterodimeric protein,” as used herein, refersto a dimer formed from two different polypeptides. A heterodimer maycomprise an anti-5T4×anti-4-1BB molecule as described herein. Aheterodimer does not include an antibody formed from four polypeptides(i.e., two light chains and two heavy chains). The terms “homodimer” or“homodimeric protein,” as used herein, refers to a dimer formed from twoidentical polypeptides.

“Fc region” or “Fc domain” refers to a polypeptide sequencecorresponding to or derived from the portion of a source antibody thatis capable of binding to Fc receptors on cells and/or the C1q componentof complement, thereby mediating the effector function of an antibody.Fc stands for “fragment crystalline,” the fragment of an antibody thatwill readily form a protein crystal. Distinct protein fragments, whichwere originally described by proteolytic digestion, can define theoverall general structure of an immunoglobulin protein. As originallydefined in the literature, the Fc region is a homodimeric proteincomprising two polypeptides that are associated by disulfide bonds, andeach comprising a hinge region, a CH2 domain, and a CH3 domain. However,more recently the term has been applied to the single chain monomercomponent consisting of CH3, CH2, and at least a portion of the hingesufficient to form a disulfide-linked dimer with a second such chain. Assuch, and depending on the context, use of the terms “Fc region” or “Fcdomain” will refer herein to either the dimeric form or the individualmonomers that associate to form the dimeric protein. For a review ofimmunoglobulin structure and function, see Putnam, The Plasma Proteins,Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol.Immunol. 31:169-217, 1994. As used herein, the term Fc includes variantsof naturally occurring sequences.

An “immunoglobulin constant region” or “constant region” is a termdefined herein to refer to a peptide or polypeptide sequence thatcorresponds to or is derived from part or all of one or more constantdomains of an immunoglobulin. In certain embodiments, the constantregion comprises IgG CH2 and CH3 domains, e.g., IgG1 CH2 and CH3domains. In certain embodiments, the constant region does not comprise aCH1 domain. In certain embodiments, the constant domains making up theconstant region are human. In some embodiments (for example, in certainvariations of a 41BB-binding polypeptide, 5T4-binding polypeptide ormultispecific polypeptides thereof), the constant region of a fusionprotein of this disclosure lacks or has minimal effector functions whileretaining the ability to bind some Fc receptors such as the neonatal Fcreceptor (FcRn) and retaining a relatively long half-life in vivo. Forexample, the constant region of a fusion protein of this disclosure donot result in, or substantially reduce the induction ofantibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependentcell-mediated phagocytosis (ADCP), complement activation, and/orcomplement-dependent cytotoxicity (CDC). In other variations, a fusionprotein of this disclosure comprises constant domains that retain one ormore effector functions, such as of one or both of ADCC and CDC. Incertain embodiments, a binding domain of this disclosure is fused to ahuman IgG1 constant region, wherein the IgG1 constant region has one ormore of the following amino acids mutated: leucine at position 234(L234), leucine at position 235 (L235), glycine at position 237 (G237),glutamate at position 318 (E318), lysine at position 320 (K320), lysineat position 322 (K322), or any combination thereof (numbering accordingto EU). For example, any one or more of these amino acids can be changedto alanine. In a further embodiment, an IgG1 Fc domain has each of L234,L235, G237, E318, K320, and K322 (according to EU numbering) mutated toan alanine (i.e., L234A, L235A, G237A, E318A, K320A, and K322A,respectively), and optionally an N297A mutation as well (i.e.,essentially eliminating glycosylation of the CH2 domain).

The terms “light chain variable region” (also referred to as “lightchain variable domain” or “V_(L)”) and “heavy chain variable region”(also referred to as “heavy chain variable domain” or “V_(H)”) refer tothe variable binding region from an antibody light and heavy chain,respectively. The variable binding regions are made up of discrete,well-defined sub-regions known as “complementarity determining regions”(CDRs) and “framework regions” (FRs). In one embodiment, the FRs arehumanized. The term “CL” refers to an “immunoglobulin light chainconstant region” or a “light chain constant region,” i.e., a constantregion from an antibody light chain. The term “CH” refers to an“immunoglobulin heavy chain constant region” or a “heavy chain constantregion,” which is further divisible, depending on the antibody isotypeinto CH1, CH2, and CH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4domains (IgE, IgM). A “Fab” (fragment antigen binding) is the part of anantibody that binds to antigens and includes the variable region and CH1domain of the heavy chain linked to the light chain via an inter-chaindisulfide bond.

As used herein, the term “linker” generally refers to a shortpolypeptide sequence connecting two sub-domains of a polypeptide.Non-limiting examples of linkers include flexible linkers comprisingglycine-serine repeats, and linkers derived from (a) an interdomainregion of a transmembrane protein (e.g., a type I transmembraneprotein); (b) a stalk region of a type II C-lectin; or (c) animmunoglobulin hinge. In some embodiments, a linker provides a spacerfunction compatible with interaction of the two sub-binding domains sothat the resulting polypeptide retains a specific binding affinity tothe same target molecule as an antibody that comprises the same lightand heavy chain variable regions. In certain embodiments, a linker iscomprised of five to about 35 amino acids, for instance, about 15 toabout 25 amino acids. As used herein, the phrase a “linker between CH3and CH1 or CL” refers to one or more amino acid residues (e.g., about2-12, about 2-10, about 4-10, about 5-10, about 6-10, about 7-10, about8-10, about 9-10, about 8-12, about 9-12, or about 10-12) between theC-terminus of a CH3 domain (e.g., a wild type CH3 or a mutated CH3) andthe N-terminus of a CH1 domain or CL domain (e.g., Cκ.

In some embodiments, depending on context, a linker may refer to (1) apolypeptide region between V_(H) and V_(L) regions in a single-chain Fv(scFv) or (2) a polypeptide region between a first binding domain and asecond binding domain in a multispecific polypeptide comprising twobinding domains. In the later example, wherein a linker connects two ormore binding domains, such a linker is referred to herein as a “bindingdomain linker.” In some embodiments, a binding domain linker maydirectly link or connect two or more binding domains, resulting in aconstruct comprising the following structure: binding domain-bindingdomain linker-binding domain. In some embodiments, the multispecificpolypeptides described herein comprise, in order from amino-terminus tocarboxyl-terminus (i) a first binding domain, (ii) a binding domainlinker, and (iii) a second binding domain. In some embodiments, amultispecific polypeptide comprises, in order from amino-terminus tocarboxyl-terminus (i) a second binding domain, (ii) a binding domainlinker, and (iii) a first binding domain. In some embodiments, a bindingdomain linker may link or connect two or more binding domains by linkingat least one binding domain to a non-binding domain polypeptide, such asan immunoglobulin Fc domain (i.e., a polypeptide comprising thestructure: Ig hinge-Ig constant region). In such embodiments, theresulting constructs may comprise the following structure: bindingdomain-Fc domain-binding domain linker-binding domain. In someembodiments, the multispecific polypeptides described herein comprise,in order from amino-terminus to carboxyl-terminus: (i) a first bindingdomain, (ii) a hinge region, (iii) an immunoglobulin constant region,(iv) a binding domain linker, and (v) a second binding domain. In someembodiments, a multispecific polypeptide comprises, in order fromamino-terminus to carboxyl-terminus (i) a second binding domain, (ii) abinding domain linker, (iii) an immunoglobulin constant region, (iv) ahinge region, and (v) a first binding domain. A polypeptide regionbetween an immunoglobulin constant region and a second binding domain ina multispecific polypeptide comprising two binding domains (e.g., abinding domain linker) may also be referred to as a “carboxyl-terminuslinker” or an “amino-terminus linker” depending on the orientation ofthe domains within the multispecific polypeptide. Non-limiting examplesof linkers are provided in Table 1.

In some embodiments, a “hinge” or a “hinge region” refers to apolypeptide derived from an immunoglobulin hinge region and locatedbetween a binding domain (e.g., a 5T4-binding domain or a 4-1BB-bindingdomain) and an immunoglobulin constant region in a polypeptide describedherein. A “wild-type immunoglobulin hinge region” refers to a naturallyoccurring upper and middle hinge amino acid sequences interposed betweenand connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) orinterposed between and connecting the CH1 and CH3 domains (for IgE andIgM) found in the heavy chain of an antibody. In certain embodiments, awild type immunoglobulin hinge region sequence is human, and cancomprise a human IgG hinge region (e.g., and IgG1, IgG2, IgG3, or IgG4hinge region).

An “altered immunoglobulin hinge region” or “variant immunoglobulinhinge region” refers to a hinge region polypeptide with one or moremutations, substitutions, insertions, or deletions compared to acorresponding parental wild-type immunoglobulin hinge region. In certainembodiments, an altered immunoglobulin hinge region is at least 70%homologous to a wild-type immunoglobulin hinge region (e.g., at least70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous). In certainembodiments, an altered immunoglobulin hinge region is a fragment of awild type immunoglobulin hinge region that has a length of about 5 aminoacids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more amino acids) up to about 120 amino acids (for instance,having a length of about 10 to about 40 amino acids or about 15 to about30 amino acids or about 15 to about 20 amino acids or about 20 to about25 amino acids). Typically, an altered immunoglobulin hinge region thatis a fragment of a wild type immunoglobulin hinge region comprises anIgG core hinge region (e.g., a polypeptide comprising the sequenceC-X-X-C, wherein X is any amino acid) as disclosed in U.S. PatentApplication Publication Nos. 2013/0129723 and 2013/0095097. Non-limitingexamples of hinges are provided in Table 1.

As used herein, the term “humanized” refers to a process of making anantibody or immunoglobulin binding proteins and polypeptides derivedfrom a non-human species (e.g., mouse or rat) less immunogenic tohumans, while still retaining antigen-binding properties of the originalantibody, using genetic engineering techniques. In some embodiments, thebinding domain(s) of an antibody or immunoglobulin binding proteins andpolypeptides (e.g., light and heavy chain variable regions, Fab, scFv)are humanized. Non-human binding domains can be humanized usingtechniques known as CDR grafting (Jones et al., Nature 321:522 (1986))and variants thereof, including “reshaping” (Verhoeyen, et al., 1988Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337;Tempest, et al., Bio/Technol 1991 9:266-271), “hyperchimerization”(Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al.,1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol148:1149-1154), and “veneering” (Mark, et al., “Derivation oftherapeutically active humanized and veneered anti-CD18 antibodies.” In:Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition totherapeutic potential. New York: Plenum Press, 1994: 291-312). Ifderived from a non-human source, other regions of the antibody orimmunoglobulin binding proteins and polypeptides, such as the hingeregion and constant region domains, can also be humanized. Knowledgeabout humanized antibodies in the art is applicable to the polypeptidesaccording to the disclosure, even if these polypeptides are notantibodies.

As used herein, the term “patient in need” refers to a patient at riskof, or suffering from, a disease, disorder or condition that is amenableto treatment or amelioration with a 5T4-binding protein or multispecificpolypeptide or a composition thereof provided herein.

As used herein, the term “pharmaceutically acceptable” refers tomolecular entities and compositions that do not generally produceallergic or other serious adverse reactions when administered usingroutes well known in the art. Molecular entities and compositionsapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans areconsidered to be “pharmaceutically acceptable.”

As used herein, the term “promoter” refers to a region of DNA involvedin binding RNA polymerase to initiate transcription.

As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or“polynucleotide” refer to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the terms encompass nucleic acids containinganalogues of natural nucleotides that have similar binding properties asthe reference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081;Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al.(1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The termnucleic acid is used interchangeably with gene, cDNA, and mRNA encodedby a gene. As used herein, the terms “nucleic acid,” “nucleic acidmolecule,” or “polynucleotide” are intended to include DNA molecules(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of theDNA or RNA generated using nucleotide analogs, and derivatives,fragments and homologs thereof.

The term “expression” refers to the biosynthesis of a product encoded bya nucleic acid. For example, in the case of nucleic acid segmentencoding a polypeptide of interest, expression involves transcription ofthe nucleic acid segment into mRNA and the translation of mRNA into oneor more polypeptides.

The terms “expression unit” and “expression cassette” are usedinterchangeably herein and denote a nucleic acid segment encoding apolypeptide of interest and capable of providing expression of thenucleic acid segment in a host cell. An expression unit typicallycomprises a transcription promoter, an open reading frame encoding thepolypeptide of interest, and a transcription terminator, all in operableconfiguration. In addition to a transcriptional promoter and terminator,an expression unit can further include other nucleic acid segments suchas, e.g., an enhancer or a polyadenylation signal.

The term “expression vector,” as used herein, refers to a nucleic acidmolecule, linear or circular, comprising one or more expression units.In addition to one or more expression units, an expression vector canalso include additional nucleic acid segments such as, for example, oneor more origins of replication or one or more selectable markers.Expression vectors are generally derived from plasmid or viral DNA, orcan contain elements of both.

As used herein, a “polypeptide,” “polypeptide chain,” or “protein”refers to a single, linear and contiguous arrangement of covalentlylinked amino acids. Polypeptides can form one or more intrachaindisulfide bonds. With regard to polypeptides as described herein,reference to modifications or alterations of amino acid residuescorresponding to those specified by SEQ ID NO includespost-translational modifications of such residues. The terms polypeptideand protein also encompass embodiments where two polypeptide chains linktogether in a non-linear fashion, such as via an interchain disulfidebond. For example, a native immunoglobulin molecule is comprised of twoheavy chain polypeptides and two light chain polypeptides. Each of theheavy chain polypeptides associate with a light chain polypeptide byvirtue of interchain disulfide bonds between the heavy and light chainpolypeptides to form two heterodimeric proteins or polypeptides (i.e., aprotein comprised of two heterologous polypeptide chains). The twoheterodimeric proteins then associate by virtue of additional interchaindisulfide bonds between the heavy chain polypeptides to form animmunoglobulin protein or polypeptide. Herein, a protein or polypeptidemay be an antibody or an antigen-binding fragment of an antibody. Insome embodiments, a protein may be an scFv-Fc-scFv protein, scFv-scFvdimer, or a diabody.

As will be appreciated by one of skill in the art, proteins andpolypeptides are defined herein in terms of the amino acid sequences ofthe individual polypeptide chains, which are indicated by the SEQ ID NOsreference throughout this disclosure. For example, in some embodimentsan scFv-Fc-scFv protein or polypeptide described herein is comprised oftwo scFv-Fc-scFv polypeptide chains associated by interchain bonds(e.g., interchain disulfide bonds) to form a dimeric scFv-Fc-scFvprotein (e.g., a homodimeric or heterodimeric scFv-Fc-scFv protein)(See, for example, FIG. 10). In such embodiments, the scFv-Fc-scFvprotein is defined by the amino acid sequences of the individualscFv-Fc-scFv polypeptide chains. Polypeptides and proteins can alsocomprise non-peptidic components, such as carbohydrate groups.Carbohydrates and other non-peptidic substituents can be added to aprotein or polypeptide by the cell in which the protein is produced, andwill vary with the type of cell. Proteins and polypeptides are definedherein in terms of their amino acid backbone structures; substituentssuch as carbohydrate groups are generally not specified, but may bepresent nonetheless.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl-terminus of thereference sequence, but is not necessarily at the carboxyl-terminus ofthe complete polypeptide.

As used herein, the term “transformation,” “transfection,” and“transduction” refer to the transfer of nucleic acid (i.e., a nucleotidepolymer) into a cell. As used herein, the term “genetic transformation”refers to the transfer and incorporation of DNA, especially recombinantDNA, into a cell. The transferred nucleic acid can be introduced into acell via an expression vector.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC,” as usedherein, refer to a cell-mediated process in which nonspecific cytotoxiccells that express FcγRs (e.g., monocytic cells such as natural killer(NK) cells and macrophages) recognize bound antibody (or other proteincapable of binding FcγRs) on a target cell and subsequently cause lysisof the target cell. In principle, any effector cell with an activatingFcγR can be triggered to mediate ADCC. The primary cells for mediatingADCC are NK cells, which express only FcγRIII, whereas monocytes,depending on their state of activation, localization, ordifferentiation, can express FcγRI, FcγRII, and FcγRIII For a review ofFcγR expression on hematopoietic cells, see, e.g., Ravetch et al., 1991,Annu. Rev. Immunol., 9:457-92.

The term “having ADCC activity,” as used herein in reference to apolypeptide or protein, means that the polypeptide or protein, forexample, one comprising an Fc domain (e.g., an immunoglobulin hingeregion and an immunoglobulin constant region having CH2 and CH3 domains)such as derived from IgG (e.g., IgG1), is capable of mediatingantibody-dependent cell-mediated cytotoxicity (ADCC) through binding ofa cytolytic Fc receptor (e.g., FcγRIII) on a cytolytic immune effectorcell expressing the Fc receptor (e.g., an NK cell). In some embodiments,a multispecific polypeptide or protein (e.g., an anti-5T4×anti-4-1BBmolecule as described herein) comprising an Fc domain may lack effectorfunction (e.g., null ADCC activity) as the result of mutations in theCH2 and/or CH3 domain.

“Complement-dependent cytotoxicity” and “CDC,” as used herein, refer toa process in which components in normal serum (“complement”), togetherwith an antibody or other C1q-complement-binding protein bound to atarget antigen, exhibit lysis of a target cell expressing the targetantigen. Complement consists of a group of serum proteins that act inconcert and in an orderly sequence to exert their effect.

The terms “classical complement pathway” and “classical complementsystem,” as used herein, are synonymous and refer to a particularpathway for the activation of complement. The classical pathway requiresantigen-antibody complexes for initiation and involves the activation,in an orderly fashion, of nine major protein components designated C1through C9. For several steps in the activation process, the product isan enzyme that catalyzes the subsequent step. This cascade providesamplification and activation of large amounts of complement by arelatively small initial signal.

The term “having CDC activity,” as used herein in reference to apolypeptide or protein, means that the polypeptide or protein, forexample, one comprising an Fc domain (e.g., an immunoglobulin hingeregion and an immunoglobulin constant region having CH2 and CH3 domains)such as derived from IgG (e.g., IgG1) is capable of mediatingcomplement-dependent cytotoxicity (CDC) through binding of C1qcomplement protein and activation of the classical complement system. Insome embodiments, a multispecific polypeptide or protein (e.g., ananti-5T4×anti-4-1BB molecule as described herein) may have lack effectorfunction (e.g., null CDC activity) as the result of one or moremutations in the CH2 and/or CH3 domains.

“Enhanced effector cell activation” as used herein, refers to theincrease, prolonging, and/or potentiation of an effector cell responseby the polypeptides or proteins described herein. In some embodiments,enhanced effector cell activation refers to an increase in the cytotoxicactivity of an effector cell. In some embodiments, enhanced effectorcell activation refers to an increase in cytokine production, cellproliferation, or a change in cell-surface molecule expression such thatthe ability of the effector cell to lyse a target cell is enhanced. Incertain embodiments, the polypeptides and proteins described hereinenhance effector cell activation by modulating Wnt/β-catenin signaling.

As used herein, the term “effector cell” refers to a cell of the immunesystem that is capable of lysing or killing a target cell, such as atumor cell. Herein, an effector cell may refer to a lymphocyte, such asa T cell, a natural killer (NK) cell, or an NKT cell, a monocyte, amacrophage, a dendritic cell, or a granulocyte. In particularembodiments, the term effector cell refers to a T cell, an NK cell, oran NKT cell.

As used herein, the terms “treatment,” “treating,” or “ameliorating”refers to either a therapeutic treatment or prophylactic/preventativetreatment. A treatment is therapeutic if at least one symptom of diseasein an individual receiving treatment improves or a treatment can delayworsening of a progressive disease in an individual, or prevent onset ofadditional associated diseases.

As used herein, the term “therapeutically effective amount (or dose)” or“effective amount (or dose)” of a polypeptide or protein describedherein or a composition thereof refers to that amount of the compoundsufficient to result in amelioration of one or more symptoms of thedisease being treated in a statistically significant manner or astatistically significant improvement in organ function. When referringto an individual active ingredient, administered alone, atherapeutically effective dose refers to that ingredient alone. Whenreferring to a combination, a therapeutically effective dose refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered serially or simultaneously (inthe same formulation or concurrently in separate formulations).

Herein, the term “statistical significance” refers the probability ofobtaining a test result that occurs by chance. For example, anobservation or test result is said to be statistically significant ifthe probability of it occurring purely by chance (p) is less than thepredetermined statistical threshold (a), or p<a. The statisticalthreshold for a particular test may be set according to thecharacteristics of the data and conventions known in the art. Forexample, a is conventionally set to 5% (0.05), such that, for a givenresult, p<0.05 in order for said result to be considered statisticallysignificant.

As used herein, a “5T4-binding domain” refers to a domain of apolypeptide described herein that specifically bind to oncofetaltrophoblast glycoprotein (5T4) (e.g., human 5T4), also known as TPBG andWnt-activated inhibitory factor 1 (WAIF1). 5T4 is a 72 kD oncofetalglycoprotein that is heavily N-glycosylated proteins with severalleucine-rich repeats associated with protein-protein interactions. Theterm “5T4” may refer to any isoform of 5T4. Exemplary human 5T4nucleotide sequences are shown in Table 1 below and provided in SEQ IDNOs: 163 and 167 and exemplary human 5T4 amino acid sequences areprovided in SEQ ID NOs: 164 and 168.

As used herein, “4-1BB-binding domain” refers to a binding domain of aprotein or polypeptide described herein that is capable of specificallybinding to human 4-1BB (also known as CD137). The term “4-1BB” may referto any isoform of 4-1BB. Exemplary human 4-1BB nucleotide sequences areshown in Table 1 below and provided in SEQ ID NOs: 161 and 165, andexemplary human 4-1BB amino acid sequences are provided in SEQ ID NOs:162 and 166.

TABLE 1 Exemplary 4-1BB and 5T4 DNA and amino acid sequences DNA AAProtein SEQ SEQ Name DNA Sequence ID AA Sequence ID FullATGGGAAACAGCTGTTACAACATAGTAGCCACTCT 161 MGNSCYNIVATLLLVLNF 162 lengthGTTGCTGGTCCTCAACTTTGAGAGGACAAGATCAT ERTRSLQDPCSNCPAGTF humanTGCAGGATCCTTGTAGTAACTGCCCAGCTGGTACA CDNNRNQICSPCPPNSFS 4-1BBTTCTGTGATAATAACAGGAATCAGATTTGCAGTCC SAGGQRTCDICRQCKGVFCTGTCCTCCAAATAGTTTCTCCAGCGCAGGTGGAC RTRKECSSTSNAECDCTPAAAGGACCTGTGACATATGCAGGCAGTGTAAAGGT GFHCLGAGCSMCEQDCKQGTTTTCAGGACCAGGAAGGAGTGTTCCTCCACCAG GQELTKKGCKDCCFGTFNCAATGCAGAGTGTGACTGCACTCCAGGGTTTCACT DQKRGICRPWTNCSLDGKGCCTGGGGGCAGGATGCAGCATGTGTGAACAGGAT SVLVNGTKERDVVCGPSPTGTAAACAAGGTCAAGAACTGACAAAAAAAGGTTG ADLSPGASSVTPPAPARETAAAGACTGTTGCTTTGGGACATTTAACGATCAGA PGHSPQIISFFLALTSTAAACGTGGCATCTGTCGACCCTGGACAAACTGTTCT LLFLLFFLTLRFSVVKRGTTGGATGGAAAGTCTGTGCTTGTGAATGGGACGAA RKKLLYIFKQPFMRPVQTGGAGAGGGACGTGGTCTGTGGACCATCTCCAGCCG TQEEDGCSCRFPEEEEGGACCTCTCTCCGGGAGCATCCTCTGTGACCCCGCCT CELGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAGAT CATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTCCGTTTC TCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAA CTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGTGA FullATGCCTGGGGGGTGCTCCCGGGGCCCCGCCGCCGG 163 MPGGCSRGPAAGDGRLRL 164 lengthGGACGGGCGTCTGCGGCTGGCGCGACTAGCGCTGG ARLALVLLGWVSSSSPTS humanTACTCCTGGGCTGGGTCTCCTCGTCTTCTCCCACC SASSFSSSAPFLASAVSA 5T4TCCTCGGCATCCTCCTTCTCCTCCTCGGCGCCGTT QPPLPDQCPALCECSEAACCTGGCTTCCGCCGTGTCCGCCCAGCCCCCGCTGC RTVKCVNRNLTEVPTDLPCGGACCAGTGCCCCGCGCTGTGCGAGTGCTCCGAG AYVRNLFLTGNQLAVLPAGCAGCGCGCACAGTCAAGTGCGTTAACCGCAATCT GAFARRPPLAELAALNLSGACCGAGGTGCCCACGGACCTGCCCGCCTACGTGC GSRLDEVRAGAFEHLPSLGCAACCTCTTCCTTACCGGCAACCAGCTGGCCGTG RQLDLSHNPLADLSPFAFCTCCCTGCCGGCGCCTTCGCCCGCCGGCCGCCGCT SGSNASVSAPSPLVELILGGCGGAGCTGGCCGCGCTCAACCTCAGCGGCAGCC NHIVPPEDERQNRSFEGMGCCTGGACGAGGTGCGCGCGGGCGCCTTCGAGCAT VVAALLAGRALQGLRRLECTGCCCAGCCTGCGCCAGCTCGACCTCAGCCACAA LASNHFLYLPRDVLAQLPCCCACTGGCCGACCTCAGTCCCTTCGCTTTCTCGG SLRHLDLSNNSLVSLTYVGCAGCAATGCCAGCGTCTCGGCCCCCAGTCCCCTT SFRNLTHLESLHLEDNALGTGGAACTGATCCTGAACCACATCGTGCCCCCTGA KVLHNGTLAELQGLPHIRAGATGAGCGGCAGAACCGGAGCTTCGAGGGCATGG VFLDNNPWVCDCHMADMVTGGTGGCGGCCCTGCTGGCGGGCCGTGCACTGCAG TWLKETEVVQGKDRLTCAGGGCTCCGCCGCTTGGAGCTGGCCAGCAACCACTT YPEKMRNRVLLELNSADLCCTTTACCTGCCGCGGGATGTGCTGGCCCAACTGC DCDPILPPSLQTSYVFLGCCAGCCTCAGGCACCTGGACTTAAGTAATAATTCG IVLALIGAIFLLVLYLNRCTGGTGAGCCTGACCTACGTGTCCTTCCGCAACCT KGIKKWMHNIRDACRDHMGACACATCTAGAAAGCCTCCACCTGGAGGACAATG EGYHYRYEINADPRLTNLCCCTCAAGGTCCTTCACAATGGCACCCTGGCTGAG SSNSDVTTGCAAGGTCTACCCCACATTAGGGTTTTCCTGGA CAACAATCCCTGGGTCTGCGACTGCCACATGGCAGACATGGTGACCTGGCTCAAGGAAACAGAGGTAGTG CAGGGCAAAGACCGGCTCACCTGTGCATATCCGGAAAAAATGAGGAATCGGGTCCTCTTGGAACTCAACA GTGCTGACCTGGACTGTGACCCGATTCTTCCCCCATCCCTGCAAACCTCTTATGTCTTCCTGGGTATTGT TTTAGCCCTGATAGGCGCTATTTTCCTCCTGGTTTTGTATTTGAACCGCAAGGGGATAAAAAAGTGGATG CATAACATCAGAGATGCCTGCAGGGATCACATGGAAGGGTATCATTACAGATATGAAATCAATGCGGACC CCAGATTAACGAACCTCAGTTCTAACTCGGATGTCTGA human ATGGGAAACAGCTGTTACAACATAGTAGCCACTCT 165 MGNSCYNIVATLLLVLNF 1664-1BB GTTGCTGGTCCTCAACTTTGAGAGGACAAGATCAT ERTRSLQDPCSNCPAGTF (ECD*)TGCAGGATCCTTGTAGTAACTGCCCAGCTGGTACA CDNNRNQICSPCPPNSFSTTCTGTGATAATAACAGGAATCAGATTTGCAGTCC SAGGQRTCDICRQCKGVFCTGTCCTCCAAATAGTTTCTCCAGCGCAGGTGGAC RTRKECSSTSNAECDCTPAAAGGACCTGTGACATATGCAGGCAGTGTAAAGGT GFHCLGAGCSMCEQDCKQGTTTTCAGGACCAGGAAGGAGTGTTCCTCCACCAG GQELTKKGCKDCCFGTFNCAATGCAGAGTGTGACTGCACTCCAGGGTTTCACT DQKRGICRPWTNCSLDGKGCCTGGGGGCAGGATGCAGCATGTGTGAACAGGAT SVLVNGTKERDVVCGPSPTGTAAACAAGGTCAAGAACTGACAAAAAAAGGTTG ADLSPGASSVTPPAPARETAAAGACTGTTGCTTTGGGACATTTAACGATCAGA PGHSPQAACGTGGCATCTGTCGACCCTGGACAAACTGTTCT TTGGATGGAAAGTCTGTGCTTGTGAATGGGACGAAGGAGAGGGACGTGGTCTGTGGACCATCTCCAGCCG ACCTCTCTCCGGGAGCATCCTCTGTGACCCCGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAG humanATGCCTGGGGGGTGCTCCCGGGGCCCCGCCGCCGG 167 MPGGCSRGPAAGDGRLRL 168 5T4GGACGGGCGTCTGCGGCTGGCGCGACTAGCGCTGG ARLALVLLGWVSSSSPTS (ECD*)TACTCCTGGGCTGGGTCTCCTCGTCTTCTCCCACC SASSFSSSAPFLASAVSATCCTCGGCATCCTCCTTCTCCTCCTCGGCGCCGTT QPPLPDQCPALCECSEAACCTGGCTTCCGCCGTGTCCGCCCAGCCCCCGCTGC RTVKCVNRNLTEVPTDLPCGGACCAGTGCCCCGCGCTGTGCGAGTGCTCCGAG AYVRNLFLTGNQLAVLPAGCAGCGCGCACAGTCAAGTGCGTTAACCGCAATCT GAFARRPPLAELAALNLSGACCGAGGTGCCCACGGACCTGCCCGCCTACGTGC GSRLDEVRAGAFEHLPSLGCAACCTCTTCCTTACCGGCAACCAGCTGGCCGTG RQLDLSHNPLADLSPFAFCTCCCTGCCGGCGCCTTCGCCCGCCGGCCGCCGCT SGSNASVSAPSPLVELILGGCGGAGCTGGCCGCGCTCAACCTCAGCGGCAGCC NHIVPPEDERQNRSFEGMGCCTGGACGAGGTGCGCGCGGGCGCCTTCGAGCAT VVAALLAGRALQGLRRLECTGCCCAGCCTGCGCCAGCTCGACCTCAGCCACAA LASNHFLYLPRDVLAQLPCCCACTGGCCGACCTCAGTCCCTTCGCTTTCTCGG SLRHLDLSNNSLVSLTYVGCAGCAATGCCAGCGTCTCGGCCCCCAGTCCCCTT SFRNLTHLESLHLEDNALGTGGAACTGATCCTGAACCACATCGTGCCCCCTGA KVLHNGTLAELQGLPHIRAGATGAGCGGCAGAACCGGAGCTTCGAGGGCATGG VFLDNNPWVCDCHMADMVTGGTGGCGGCCCTGCTGGCGGGCCGTGCACTGCAG TWLKETEVVQGKDRLTCAGGGCTCCGCCGCTTGGAGCTGGCCAGCAACCACTT YPEKMRNRVLLELNSADLCCTTTACCTGCCGCGGGATGTGCTGGCCCAACTGC DCDPILPPSLQTSCCAGCCTCAGGCACCTGGACTTAAGTAATAATTCG CTGGTGAGCCTGACCTACGTGTCCTTCCGCAACCTGACACATCTAGAAAGCCTCCACCTGGAGGACAATG CCCTCAAGGTCCTTCACAATGGCACCCTGGCTGAGTTGCAAGGTCTACCCCACATTAGGGTTTTCCTGGA CAACAATCCCTGGGTCTGCGACTGCCACATGGCAGACATGGTGACCTGGCTCAAGGAAACAGAGGTAGTG CAGGGCAAAGACCGGCTCACCTGTGCATATCCGGAAAAAATGAGGAATCGGGTCCTCTTGGAACTCAACA GTGCTGACCTGGACTGTGACCCGATTCTTCCCCCATCCCTGCAAACCTCT *ECD = Extracellular domain

As used herein, a “multispecific polypeptide” refers to a polypeptidecomprising two or more binding domains each capable of specificallybinding to a target antigen. For example, the polypeptides describedherein may comprise 2, 3, 4, or more binding domains and may be able tobind 2, 3, 4, or more target antigens. In some embodiments, amultispecific polypeptide is a bispecific polypeptide. Herein, a“bispecific polypeptide” comprises two binding domains and capable ofbinding to two distinct target antigens. In some embodiments, thebispecific polypeptides described herein comprise a first binding domainthat specifically binds to a cell surface antigen expressed on a targetcell, such as 5T4. In some embodiments, the bispecific polypeptidesdescribed herein comprise a binding domain that specifically binds to acell surface antigen expressed on an effector cell, such as 4-1BB. Inparticular embodiments, the multispecific polypeptide is an ADAPTIR™homodimer bispecific polypeptide in the format scFv-Fc-scFv.

Multispecific polypeptides using scaffolds are disclosed, for instance,in PCT Publication Nos. WO 2007/146968; WO 2010/040105; WO 2010/003108;WO 2016/094873; WO 2017/053469; U.S. Patent Application Publication No.2006/0051844; and U.S. Pat. Nos. 7,166,707; and 8,409,577, which areeach incorporated herein by reference in their entirety. In certainembodiments, the multispecific polypeptides described herein arebispecific polypeptides and may comprise an scFv-Fc-scFv structure, alsoreferred to herein as an ADAPTIR™ polypeptide, or Format 1, an exemplaryembodiment of which is shown in FIG. 10. The structure of a polypeptidecomprising a Format 1 structure comprises, from N-terminus toC-terminus: a first scFv binding domain-an immunoglobulin (Ig) hingeregion-an Ig constant region-a second scFv binding domain (FIG. 10). Incertain embodiments, the multispecific polypeptides described herein maycomprise an IgG-scFv structure (also referred to herein as the Morrisonformat, or Format 2). The structure of a polypeptide comprising a Format2 structure comprises, from N-terminus to C-terminus: an scFv bindingdomain-an Ig constant region-an Ig hinge region-an Ig variable region.Format 2 is, essentially, an intact Ig molecule comprising a C-terminalscFv domain.

Binding Polypeptides and Proteins

In some embodiments, the present disclosure describes polypeptidescapable of specifically binding to 5T4 (e.g., 5T4-binding polypeptides),as well as multispecific polypeptides and proteins comprising thesebinding domains. Such embodiments may be referred to as 5T4-bindingpolypeptides. In particular embodiments, the present invention providesmultispecific binding proteins comprising a 5T4-binding domain and asecond binding domain capable of binding to a cell-surface molecule onan effector cell. In particular embodiments, the present inventionprovides bispecific binding proteins comprising a 5T4-binding domain anda second binding domain capable of binding to 4-1BB (e.g., a4-1BB-binding domain). In some embodiments, the 5T4-binding polypeptidescomprise the structure, from N-terminus to C-terminus: a 5T4-bindingdomain-Fc domain.

In some embodiments, the present disclosure describes polypeptidescapable of specifically binding to 4-1BB, as well as multispecificpolypeptides and proteins comprising these binding domains. Suchembodiments may be referred to as 4-1BB-binding polypeptides. Inparticular embodiments, the present invention provides multispecificpolypeptides comprising a 4-1BB-binding domain and a second bindingdomain capable of binding to a cell-surface molecule on a target cell.In particular embodiments, the present invention provides bispecificpolypeptides comprising a 4-1BB-binding domain and a second bindingdomain capable of binding to 5T4 (e.g., a 5T4-binding domain). In someembodiments, the 4-1BB-binding polypeptides comprise the structure, fromN-terminus to C-terminus: a 4-1BB-binding domain-Fc domain.

In some embodiments, the polypeptides described herein can furthercomprise immunoglobulin constant regions, linker peptides, hingeregions, immunoglobulin dimerization/heterodimerization domains,junctional amino acids, tags, etc. These components of the disclosedpolypeptides and proteins are described in further detail below.

The polypeptides and proteins described herein (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) comprise one or more binding domains capable ofspecifically binding a target antigen. The binding domains describedherein can be in the form of an antibody or a fusion protein of any of avariety of different formats (e.g., the fusion protein can be in theform of a bispecific or multispecific molecule). Non-limiting examplesof bi specific molecules include an scFv-Fc-scFv molecule (e.g. abispecific protein comprising the structure of Format 1), an scFv-Igmolecule (e.g. a bispecific protein comprising the structure of Format2) and an scFv-scFv molecule. In some embodiments, the bispecificmolecules described herein comprise or consist of a first binding domainscFv linked to a second binding domain scFv and do not include othersequences such as an immunoglobulin constant region. In otherembodiments, the bispecific protein described herein are diabodies.

In some embodiments, the polypeptides and proteins described herein areconjugated to a drug or a toxic moiety.

In some embodiments, the polypeptides and proteins described herein(e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) may comprise sequences aminoacid and/or nucleic acid shown in Tables 2-10. In certain embodiments,the binding domains of the polypeptides described herein comprise (i) animmunoglobulin light chain variable region (V_(L)) comprising CDRsLCDR1, LCDR2, and LCDR3, and (ii) an immunoglobulin heavy chain variableregion (V_(H)) comprising CDRs HCDR1, HCDR2, and HCDR3. In someembodiments, amino acid sequences provided for polypeptide constructs donot include the human immunoglobulin leader sequences. CDR sequences andamino acid substitution positions shown are those defined using the IMGTcriteria (Brochet et al, Nucl. Acids Res. (2008) 36, W503-508). In somecases, the sequences shown in the disclosure, including in Tables 2-10,contain amino acid substitutions relative to a parent sequence (e.g. ananti-4-1BB antibody or binding fragment thereof such as clone 1618/1619,which is disclosed in PCT Application Publication No. WO 2016/185016, oran anti-5T4 antibody or binding fragment thereof such as those disclosedin PCT Application Publication No. WO 2016/185016). In some embodiments,the parent sequence of an anti-5T4 binding domain comprises the aminoacid sequence provided in SEQ ID NOs: 38 and 68. In some embodiments,the parent sequence of an anti-4-1BB binding domain comprises the aminoacid sequence provided in SEQ ID NOs: 28 and 16. Exemplary parentalsequences for the anti-4-1BB and anti-5T4 binding domains describedherein are shown below in Table 2. Underlined text indicates CDRsequences.

TABLE 2 Exemplary Binding Domain Parental Sequences Parental SEQ IDSequence Amino Acid Sequence NO: 1618EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGS 28 anti-4-GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDY 1BB V_(H)WGQGTLVTVSS 1618 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQ16 anti-4- SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK1BB V_(L) 1210 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSG38 anti-5T4 GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYV_(H) WGQGTLVTVSS 1210 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQ 68 anti-5T4SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK V_(L)

In certain embodiments, a binding domain V_(L) and/or V_(H) region ofthe present disclosure is derived from a V_(L) and/or V_(H) of a parentV_(L) and/or V_(H) region (e.g., 1618/1619 as described in PCTApplication Publication No. WO 2016/185016) and optionally containsabout one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions,about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions,about one or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acidsubstitutions (e.g., conservative amino acid substitutions ornon-conservative amino acid substitutions), or a combination of theabove-noted changes, when compared to the V_(L) and/or V_(H) sequence ofa known monoclonal antibody. The insertion(s), deletion(s) orsubstitution(s) can be anywhere in the V_(L) and/or V_(H) region,including at the amino- or carboxyl-terminus or both ends of thisregion, provided that each CDR comprises zero changes or at most one,two, or three changes. In some embodiments, the binding domaincontaining the modified V_(L) and/or V_(H) region can still specificallybind its target with an affinity similar to or greater than the parentbinding domain.

In some embodiments, a binding domain described herein is derived froman antibody and comprises a variable heavy chain (V_(H)) and a variablelight chain (V_(L)). For example, an scFv comprising a V_(H) and V_(L)chain. These binding domains and variable chains may be arranged in anyorder that still retains some binding to the target(s). For example, thevariable domains may be arranged in the order such as V_(H) 5T4-V_(L)5T4-V_(H) 4-1BB-V_(L) 4-1BB; V_(L) 5T4-V_(H) 5T4-V_(H) 4-1BB-V_(L)4-1BB; V_(H) 5T4-V_(L) 5T4-V_(L) 4-1BB-V_(H) 4-1BB; V_(L) 5T4-V_(H)5T4-V_(L) 4-1BB-V_(H) 4-1BB; V_(H) 4-1BB-V_(L) 4-1BB-V_(H) 5T4-V_(L)5T4; V_(L) 4-1BB-V_(H) 4-1BB-V_(L) 5T4-V_(H) 5T4; V_(H) 4-1BB-V_(L)4-1BB -V_(L) 5T4-V_(H) 5T4; or V_(L) 4-1BB-V_(H) 4-1BB-V_(H) 5T4-V_(L)5T4. The pairs of V_(H) regions and V_(L) regions in the binding domainbinding to 4-1BB and/or the binding domain binding to 5T4 may be in theformat of a single chain antibody (scFv).

In some embodiments, the polypeptides and proteins described herein(e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) comprise binding domains thatare scFvs. In such embodiments, the binding domains may be referred toas scFv domains. In some embodiments, a binding domain is a single-chainFv fragment (scFv) that comprises V_(H) and V_(L) regions specific for atarget of interest. In certain embodiments, the V_(H) and V_(L) regionsare human or humanized. In some variations, a binding domain is asingle-chain Fv (scFv) comprising V_(L) and V_(H) regions joined by apeptide linker.

The use of peptide linkers for joining V_(L) and V_(H) regions iswell-known in the art, and a large number of publications exist withinthis particular field. In some embodiments, a peptide linker is a 15merconsisting of three repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence((Gly₄Ser)₃) (SEQ ID NO: 96). Other linkers have been used, and phagedisplay technology, as well as selective infective phage technology, hasbeen used to diversify and select appropriate linker sequences (Tang etal., J. Biol. Chem. 271, 15682-15686, 1996; Hennecke et al., ProteinEng. 11, 405-410, 1998). In certain embodiments, the V_(L) and V_(H)regions are joined by a peptide linker having an amino acid sequencecomprising the formula (Gly₄Ser)_(n), wherein n=1-5. For instance, inone embodiment of the invention, the linker comprises (Gly4Ser)₄. Othersuitable linkers can be obtained by optimizing a simple linker (e.g.,(Gly₄Ser)_(n)) through random mutagenesis. In some embodiments, theV_(H) region of the scFv described herein may be positioned N-terminallyto a linker sequence. In some embodiments, the V_(L) region of the scFvsdescribed herein may be positioned C-terminally to the linker sequence.In some embodiments, the scFv may bind to 5T4 and/or 4-1BB moreeffectively than the antibody comprising the same V_(H) and V_(L) regionsequences in the same orientation. In certain embodiments, the scFv maybind more effectively to 5T4 and/or 4-1BB in the V_(L)-V_(H) orientationthan in the V_(H)-V_(L) orientation, or vice versa.

In some embodiments, the polypeptide comprises a binding domain linkerlinking the binding domains (e.g., linking the scFv domains). In someembodiments, the binding domain linker is a Gly₄Ser linker. In someembodiments, the binding domain linker is a 20mer consisting of fourrepeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence ((Gly₄Ser)₃) (SEQID NO: 98). In some embodiments, the binding domain linker comprises anamino acid sequence selected from SEQ ID NOs 86-108. Other linkers havebeen used, and phage display technology, as well as selective infectivephage technology, has been used to diversify and select appropriatelinker sequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996;Hennecke et al., Protein Eng. 11, 405-410, 1998). In certainembodiments, the V_(L) and V_(H) regions are joined by a peptide linkerhaving an amino acid sequence comprising the formula (Gly₄Ser)_(n),wherein n=1-5. Other suitable linkers can be obtained by optimizing asimple linker (e.g., (Gly₄Ser)_(n)) through random mutagenesis. In someinstances, a bispecific molecule may comprise an scFv binding to 5T4linked to an scFv binding to 4-1BB. In some embodiments, bispecificmolecules do not comprise a hinge region or a constant region.

In some embodiments, the polypeptides and proteins described herein(e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) further comprise a hinge. Insome embodiments, the hinge is an altered immunoglobulin hinge in whichone or more cysteine residues in a wild type immunoglobulin hinge regionis substituted with one or more other amino acid residues (e.g., serineor alanine). Exemplary altered immunoglobulin hinges, carboxyl-terminuslinkers, and amino-terminus linkers include an immunoglobulin human IgG1hinge region having one, two or three cysteine residues found in a wildtype human IgG1 hinge substituted by one, two or three different aminoacid residues (e.g., serine or alanine). An altered immunoglobulin hingecan additionally have a proline substituted with another amino acid(e.g., serine or alanine). For example, the above-described alteredhuman IgG1 hinge can additionally have a proline locatedcarboxyl-terminal to the three cysteines of wild type human IgG1 hingeregion substituted by another amino acid residue (e.g., serine,alanine). In one embodiment, the prolines of the core hinge region arenot substituted. In certain embodiments, a hinge, a carboxyl-terminuslinker, or an amino-terminus linker polypeptide comprises or is asequence that is at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to a wild type immunoglobulin hinge region, such asa wild type human IgG1 hinge, a wild type human IgG2 hinge, or a wildtype human IgG4 hinge.

In certain embodiments, a hinge present in a polypeptide that forms aheterodimer with another polypeptide chain can be an immunoglobulinhinge, such as a wild-type immunoglobulin hinge region or an alteredimmunoglobulin hinge region thereof. In certain embodiments, a hinge ofone polypeptide chain of a heterodimeric protein is identical to acorresponding hinge of the other polypeptide chain of the heterodimer.In certain other embodiments, a hinge of one chain is different fromthat of the other chain (in their length or sequence). The differenthinges in the different chains allow different manipulation of thebinding affinities of the binding domains to which the hinges areconnected, so that the heterodimer is able to preferentially bind to thetarget of one binding domain over the target of the other bindingdomain. For example, in certain embodiments, a heterodimeric protein hasa 4-1BB-binding domain in one chain and a 5T4-binding domain in anotherchain. Having two different hinges in the two chains may allow theheterodimer to bind to the 5T4 first, and then to 4-1BB second. Thus,the heterodimer may recruit 4-1BB-expressing effector cells to5T4-expressing target cells (e.g., 5T4-expressing tumor or cancercells), which in turn may damage or destroy the 5T4-expressing cells.

In certain embodiments, a carboxyl-terminus linker or an amino-terminuslinker is a flexible linker sequence comprising glycine-serine (e.g.,Gly₄Ser) repeats. In certain embodiments, the linker comprises threeGly₄Ser repeats (SEQ ID NO: 96) followed by a proline residue. Incertain embodiments the proline residue is followed by an amino acidselected from the group consisting of glycine, arginine and serine. Insome embodiments, a carboxyl-terminus linker or an amino-terminus linkercomprises or consists of a sequence selected from SEQ ID NO: 86-108.

Some exemplary hinge, carboxyl-terminus linker, and amino-terminuslinker sequences suitable for use in accordance with the presentdisclosure are Table 3 below. Additional exemplary hinge and linkerregions are set forth in SEQ ID NOs: 241-244, 601, 78, 763-791, 228,379-434, 618-749 of U.S. Patent Publication No. 2013/0129723 (saidsequences incorporated by reference herein).

TABLE 3 Exemplary hinges and linkers Hinge Region Amino Acid SequenceSEQ ID sss(s)-hIgG1 hinge EPKSSDKTHTSPPSS  71 csc(s)-hIgG1 hingeEPKSCDKTHTSPPCS  72 ssc(s)-hIgG1 hinge EPKSSDKTHTSPPCS  73scc(s)-hIgG1 hinge EPKSSDKTHTCPPCS  74 css(s)-hIgG1 hingeEPKSCDKTHTSPPSS  75 scs(s)-hIgG1 hinge EPKSSDKTHTCPPSS  76ccc(s)-hIgG1 hinge EPKSCDKTHTSPPCS  77 ccc(p)-hIgG1 hingeEPKSCDKTHTSPPCP  78 sss(p)-hIgG1 hinge EPKSSDKTHTSPPSP  79csc(p)-hIgG1 hinge EPKSCDKTHTSPPCP  80 ssc(p)-hIgG1 hingeEPKSSDKTHTSPPCP  81 scc(p)-hIgG1 hinge EPKSSDKTHTCPPCP  82css(p)-hIgG1 hinge EPKSCDKTHTSPPSP  83 scs(p)-hIgG1 hingeEPKSSDKTHTCPPSP  84 Scppcp SCPPCP  85 STD1 NYGGGGSGGGGSGGGGSGNS  86 STD2NYGGGGSGGGGSGGGGSGNYGGGGSGGGGSGGGGSGNS  87 H1 NS  88 H2 GGGGSGNS  89 H3NYGGGGSGNS  90 H4 GGGGSGGGGSGNS  91 H5 NYGGGGSGGGGSGNS  92 H6GGGGSGGGGSGGGGSGNS  93 H7 GCPPCPNS  94 Gly₄Ser GGGGS  95 (G₄S)₃GGGGSGGGGSGGGGS  96 H105 SGGGGSGGGGSGGGGS  97 (G₄S)₄GGGGSGGGGSGGGGSGGGGS  98 H75 (NKG2A quadruple mutant)QRHNNSSLNTGTQMAGHSPNS  99 H83 (NKG2A derived) SSLNTGTQMAGHSPNS 100H106 (NKG2A derived) QRHNNSSLNTGTQMAGHS 101 H81 (NKG2D derived)EVQIPLTESYSPNS 102 H91 (NKG2D derived) NSLANQEVQIPLTESYSPNS 103 H94SGGGGSGGGGSGGGGSPNS 104 H111 SGGGGSGGGGSGGGGSPGS 105 H113SGGGGSGGGGSGGGGSPAS 106 H114 SGGGGSGGGGSGGGGSPS 107 H115SGGGGSGGGGSGGGGSPSS 108

In other embodiments, the polypeptides and proteins described hereininclude a heterodimerization domain that is capable ofheterodimerization with a different heterodimerization domain in asecond, non-identical polypeptide chain. In certain variations, thesecond polypeptide chain for heterodimerization includes a secondbinding domain. Accordingly, in certain embodiments of the presentdisclosure, two non-identical polypeptide chains, one comprising thepolypeptide comprising a first binding domain and the second optionallycomprising a second binding domain, dimerize to form a heterodimericbinding protein. Dimerization/heterodimerization domains can be usedwhere it is desired to form heterodimers from two non-identicalpolypeptide chains, where one or both polypeptide chains comprise abinding domain. In certain embodiments, one polypeptide chain member ofcertain heterodimers described herein does not contain a binding domain.Examples of types of heterodimers include those described in U.S. PatentApplication Publication Nos. 2013/0095097 and 2013/0129723, andInternational PCT Publication No. WO 2016/094873.

In certain embodiments, the first and second polypeptide chains dimerizevia the inclusion of an “immunoglobulin dimerization domain” or“immunoglobulin heterodimerization domain.” An “immunoglobulindimerization domain” or “immunoglobulin heterodimerization domain”refers herein to an immunoglobulin domain of a first polypeptide chainthat preferentially interacts or associates with a differentimmunoglobulin domain of a second polypeptide chain, wherein theinteraction of the different immunoglobulin domains substantiallycontributes to or efficiently promotes heterodimerization of the firstand second polypeptide chains (i.e., the formation of a dimer betweentwo different polypeptide chains, which is also referred to as a“heterodimer”). The immunoglobulin heterodimerization domains in thepolypeptide chains of a heterodimer are different from each other andthus can be differentially modified to facilitate heterodimerization ofboth chains and to minimize homodimerization of either chain.Immunoglobulin heterodimerization domains provided herein allow forefficient heterodimerization between different polypeptides andfacilitate purification of the resulting heterodimeric protein.

As provided herein, immunoglobulin heterodimerization domains useful forpromoting heterodimerization of two different polypeptide chainsaccording to the present disclosure include wild-type and alteredimmunoglobulin CH1 and CL domains, for instance, human CH1 and CLdomains. In certain embodiments, an immunoglobulin heterodimerizationdomain is a wild-type CH1 domain, such as a wild type IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, IgD, IgE, or IgM CH1 domain, for example, as set forthin SEQ ID NOs: 114, 186-192 and 194, respectively, of U.S. PatentApplication Publication No. 2013/0129723 or SEQ ID NO: 114 of U.S.Patent Application Publication No. 2013/0129723 (said sequenceincorporated by reference herein). In further embodiments, a cysteineresidue of a wild-type CH1 domain (e.g., a human CH1) involved informing a disulfide bond with a wild type immunoglobulin CL domain(e.g., a human CL) is deleted or substituted in the alteredimmunoglobulin CH1 domain such that a disulfide bond is not formedbetween the altered CH1 domain and the wild-type CL domain.

In certain embodiments, an immunoglobulin heterodimerization domain is awild-type CL domain, such as a wild type Cκ domain or a wild type Cλdomain, for example, as set forth in SEQ ID NOs: 112 and 113,respectively, of U.S. Patent Application Publication No. 2013/0129723(said sequences incorporated by reference herein). In furtherembodiments, an immunoglobulin heterodimerization domain is an alteredimmunoglobulin CL domain, such as an altered Cκ or Cλ domain, forinstance, an altered human Cκ or human Cλ domain. In certainembodiments, a cysteine residue of a wild-type CL domain involved informing a disulfide bond with a wild type immunoglobulin CH1 domain isdeleted or substituted in the altered immunoglobulin CL domain, forexample a Cκ domain as set forth in SEQ ID NO: 141 of U.S. PatentApplication Publication No. 2013/0129723 or a Cλ domain as set forth inSEQ ID NO: 140 of U.S. Patent Application Publication No. 2013/0129723(said sequences incorporated by reference herein). In certainembodiments, only the last cysteine of the wild type human Cκ domain isdeleted in the altered Cκ domain because the first arginine deleted fromthe wild type human Cκ domain can be provided by a linker that has anarginine at its carboxyl-terminus and links the amino-terminus of thealtered Cκ domain with another domain (e.g., an immunoglobulinsub-region, such as a sub-region comprising immunoglobulin CH2 and CH3domains).

In further embodiments, an immunoglobulin heterodimerization domain isan altered Cκ domain that contains one or more amino acid substitutions,as compared to a wild type Cκ domain, at positions that may be involvedin forming the interchain-hydrogen bond network at a Cκ-Cκ interface.For example, in certain embodiments, an immunoglobulinheterodimerization domain is an altered human Cκ domain having one ormore amino acids at positions N29, N30, Q52, V55, T56, S68 or T70 thatare substituted with a different amino acid. The numbering of the aminoacids is based on their positions in the altered human Cκ sequence asset forth in SEQ ID NO: 141 of U.S. Patent Application Publication No.2013/0129723 (said sequence incorporated by reference herein). Incertain embodiments, an immunoglobulin heterodimerization domain is analtered human Cκ domain having one, two, three or four amino acidsubstitutions at positions N29, N30, V55, or T70. The amino acid used asa substitute at the above-noted positions can be an alanine, or an aminoacid residue with a bulk side chain moiety such as arginine, tryptophan,tyrosine, glutamate, glutamine, lysine aspartate, methionine, serine orphenylalanine. Altered human Cκ domains are those that facilitateheterodimerization with a CH1 domain, but minimize homodimerization withanother Cκ domain. Representative altered human Cκ domains are set forthin SEQ ID NOs: 142-178 of U.S. Patent Application Publication No.2013/0129723; SEQ ID NOs: 160 (N29W V55A T70A), 161 (N29Y V55A T70A),202 (T70E N29A N30A V55A), 167 (N30R V55A T70A), 168 (N30K V55A T70A),170 (N30E V55A T70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E),176 (N29Y N30Y V55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E),838 (N30D V55A T70E), 839 (N30M V55A T70E), 840 (N305 V55A T70E), and841 (N30F V55A T70E) of U.S. Patent Application Publication No.2013/0129723 (said sequences incorporated by reference herein).

In certain embodiments, in addition to or alternative to the mutationsin Cκ domains described herein, both the immunoglobulinheterodimerization domains (i.e., immunoglobulin CH1 and CL domains) ofa polypeptide heterodimer have mutations so that the resultingimmunoglobulin heterodimerization domains form salt bridges (i.e., ionicinteractions) between the amino acid residues at the mutated sites. Forexample, the immunoglobulin heterodimerization domains of a polypeptideheterodimer can be a mutated CH1 domain in combination with a mutated Cκdomain. In the mutated CH1 domain, valine at position 68 (V68) of thewild type human CH1 domain is substituted by an amino acid residuehaving a negative charge (e.g., aspartate or glutamate), whereas leucineat position 29 (L29) of a mutated human Cκ domain in which the firstarginine and the last cysteine have been deleted is substituted by anamino acid residue having a positive charge (e.g., lysine, arginine orhistidine). The charge-charge interaction between the amino acid residuehaving a negative charge of the resulting mutated CH1 domain and theamino acid residue having a positive charge of the resulting mutated Cκdomain forms a salt bridge, which stabilizes the heterodimeric interfacebetween the mutated CH1 and Cκ domains. Alternatively, V68 of the wildtype CH1 can be substituted by an amino acid residue having a positivecharge, whereas L29 of a mutated human Cκ domain in which the firstarginine and the last cysteine have been deleted can be substituted byan amino acid residue having a negative charge. Exemplary mutated CH1sequences in which V68 is substituted by an amino acid with either anegative or positive charge are set forth in SEQ ID NOs: 844 and 845 ofU.S. Patent Application Publication No. 2013/0129723 (said sequencesincorporated by reference herein). Exemplary mutated Cκ sequences inwhich L29 is substituted by an amino acid with either a negative orpositive charge are set forth in SEQ ID NOs: 842 and 843 of U.S. PatentApplication Publication No. 2013/0129723 (said sequences incorporated byreference herein).

Positions other than V68 of human CH1 domain and L29 of human Cκ domaincan be substituted with amino acids having opposite charges to produceionic interactions between the amino acids in addition or alternative tothe mutations in V68 of CH1 domain and L29 of Cκ domain. Such positionscan be identified by any suitable method, including random mutagenesis,analysis of the crystal structure of the CH1-Cκ pair to identify aminoacid residues at the CH1-Cκ interface, and further identifying suitablepositions among the amino acid residues at the CH1-Cκ interface using aset of criteria (e.g., propensity to engage in ionic interactions,proximity to a potential partner residue, etc.).

In certain embodiments, polypeptide heterodimers of the presentdisclosure contain only one pair of immunoglobulin heterodimerizationdomains. For example, a first chain of a polypeptide heterodimer cancomprise a CH1 domain as an immunoglobulin heterodimerization domain,while a second chain can comprise a CL domain (e.g., a Cκ or Cλ) as animmunoglobulin heterodimerization domain. Alternatively, a first chaincan comprise a CL domain (e.g., a Cκ or Cλ) as an immunoglobulinheterodimerization domain, while a second chain can comprise a CH1domain as an immunoglobulin heterodimerization domain. As set forthherein, the immunoglobulin heterodimerization domains of the first andsecond chains are capable of associating to form a heterodimeric proteinof this disclosure.

In certain other embodiments, heterodimeric proteins of the presentdisclosure can have two pairs of immunoglobulin heterodimerizationdomains. For example, a first chain of a heterodimer can comprise twoCH1 domains, while a second chain can have two CL domains that associatewith the two CH1 domains in the first chain. Alternatively, a firstchain can comprise two CL domains, while a second chain can have two CH1domains that associate with the two CL domains in the first chain. Incertain embodiments, a first polypeptide chain comprises a CH1 domainand a CL domain, while a second polypeptide chain comprises a CL domainand a CH1 domain that associate with the CH1 domain and the CL domain,respectively, of the first polypeptide chain.

In the embodiments where a heterodimeric protein comprises only oneheterodimerization pair (i.e., one immunoglobulin heterodimerizationdomain in each chain), the immunoglobulin heterodimerization domain ofeach chain can be located amino-terminal to the immunoglobulin constantregion of that chain. Alternatively, the immunoglobulinheterodimerization domain in each chain can be located carboxyl-terminalto the immunoglobulin constant region of that chain.

In the embodiments where a heterodimeric protein comprises twoheterodimerization pairs (i.e., two immunoglobulin heterodimerizationdomains in each chain), both immunoglobulin heterodimerization domainsin each chain can be located amino-terminal to the immunoglobulinconstant region of that chain. Alternatively, both immunoglobulinheterodimerization domains in each chain can be locatedcarboxyl-terminal to the immunoglobulin constant region of that chain.In further embodiments, one immunoglobulin heterodimerization domain ineach chain can be located amino-terminal to the immunoglobulin constantregion of that chain, while the other immunoglobulin heterodimerizationdomain of each chain can be located carboxyl-terminal to theimmunoglobulin constant region of that chain. In other words, in thoseembodiments, the immunoglobulin constant region is interposed betweenthe two immunoglobulin heterodimerization domains of each chain.

As indicated herein, in some embodiments, the polypeptides and proteins(e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) of the present disclosurefurther comprise an immunoglobulin constant region (also referred toherein as a constant region, Fc domain, Fc region, and the like) in apolypeptide chain. In some embodiments, the immunoglobulin constantregion comprises an amino acid sequence according to SEQ ID NO: 158,160, or a variant thereof. The inclusion of an immunoglobulin constantregion slows clearance of the polypeptides and proteins of the presentinvention from circulation after administration to a subject. Bymutations or other alterations, an immunoglobulin constant regionfurther enables relatively easy modulation of polypeptide effectorfunctions (e.g., ADCC, ADCP, CDC, complement fixation, and binding to Fcreceptors), which can either be increased or decreased depending on thedisease being treated, as known in the art and described herein. Incertain embodiments, the polypeptides and proteins described hereincomprise an immunoglobulin constant region capable of mediating one ormore of these effector functions. In other embodiments, one or more ofthese effector functions are reduced or absent in an immunoglobulinconstant region of a polypeptide or protein described herein presentdisclosure, as compared to a corresponding wild-type immunoglobulinconstant region. For example, for dimeric 5T4-binding or 4-1BB-bindingpolypeptides designed to enhance effector cell activation, such as,e.g., via the inclusion of a 4-1BB-binding domain, an immunoglobulinconstant region preferably has reduced or no effector function relativeto a corresponding wild-type immunoglobulin constant region.

An immunoglobulin constant region present in the polypeptides andproteins of the present disclosure (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof) can comprise or can be derived from part or all of: a CH2domain, a CH3 domain, a CH4 domain, or any combination thereof. Forexample, an immunoglobulin constant region can comprise a CH2 domain, aCH3 domain, both CH2 and CH3 domains, both CH3 and CH4 domains, two CH3domains, a CH4 domain, two CH4 domains, and a CH2 domain and part of aCH3 domain. In certain embodiments, the polypeptides or proteinsdescribed herein do not comprise a CH1 domain.

A polypeptide or protein described herein may comprise a wild typeimmunoglobulin CH2 domain or an altered immunoglobulin CH2 domain fromcertain immunoglobulin classes or subclasses (e.g., IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, or IgD) and from various species (including human,mouse, rat, and other mammals). In certain embodiments, a CH2 domain ofa polypeptide or a protein described herein is a wild type humanimmunoglobulin CH2 domain, such as wild type CH2 domains of human IgG1,IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, as set forth in SEQ ID NOs: 115,199-201 and 195-197, respectively, of U.S. Patent ApplicationPublication No. 2013/0129723 (said sequences incorporated by referenceherein). In certain embodiments, the CH2 domain is a wild type humanIgG1 CH2 domain as set forth in SEQ ID NO: 115 of U.S. PatentApplication Publication No. US 2013/0129723 (said sequence incorporatedby reference herein).

In certain embodiments, an altered CH2 region in a polypeptide or aprotein of the present disclosure comprises or is a sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to a wild type immunoglobulin CH2 region, such as the CH2region of wild type human IgG1, IgG2, or IgG4, or mouse IgG2a (e.g.,IGHG2c).

An altered immunoglobulin CH2 region in a polypeptide or protein of thepresent disclosure (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof) can bederived from a CH2 region of various immunoglobulin isotypes, such asIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgD, from various species(including human, mouse, rat, and other mammals). In certainembodiments, an altered immunoglobulin CH2 region in a fusion protein ofthe present disclosure can be derived from a CH2 region of human IgG1,IgG2 or IgG4, or mouse IgG2a (e.g., IGHG2c), whose sequences are setforth in SEQ ID NOs: 115, 199, 201, and 320 of U.S. Patent ApplicationPublication No. 2013/0129723 (said sequences incorporated by referenceherein). In certain embodiments, an altered CH2 domain of a polypeptideor a protein described herein (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof) is an altered human IgG1 CH2 domain with mutations known in theart that enhance or reduce immunological activities (i.e., effectorfunctions) such as ADCC, ADCP, CDC, complement fixation, Fc receptorbinding, or any combination thereof.

In certain embodiments, a CH2 domain of a polypeptide or a proteindescribed herein is an altered immunoglobulin CH2 region (e.g., analtered human IgG1 CH2 domain) that comprises one or more amino aciddeletions or substitutions. In some embodiments, the CH2 domaincomprises an amino acid substitution at the asparagine of position 297(e.g., asparagine to alanine). Such an amino acid substitution reducesor eliminates glycosylation at this site and abrogates efficient Fcbinding to FcγR and C1q. The sequence of an altered human IgG1 CH2domain with an Asn to Ala substitution at position 297 is set forth inSEQ ID NO: 324 of U.S. Patent Application Publication No. 2013/0129723(said sequence incorporated by reference herein). In some embodiments,the altered CH2 domain comprises at least one substitution or deletionat positions 234 to 238. For example, an immunoglobulin CH2 region cancomprise a substitution at position 234, 235, 236, 237 or 238; positions234 and 235; positions 234 and 236; positions 234 and 237; positions 234and 238; positions 234-236; positions 234, 235 and 237; positions 234,236 and 238; positions 234, 235, 237, and 238; positions 236-238; or anyother combination of two, three, four, or five amino acids at positions234-238. In some embodiments, an altered CH2 region comprises one ormore (e.g., two, three, four or five) amino acid deletions at positions234-238, for instance, at one of position 236 or position 237 while theother position is substituted. In certain embodiments, the amino acidresidues at one or more of positions 234-238 has been replaced with oneor more alanine residues. In further embodiments, only one of the aminoacid residues at positions 234-238 have been deleted while one or moreof the remaining amino acids at positions 234-238 can be substitutedwith another amino acid (e.g., alanine or serine).

In some embodiments, the above-noted mutation(s) decrease or eliminatethe ADCC activity or Fc receptor-binding capability of a polypeptidethat comprises the altered CH2 domain.

In certain other embodiments, a CH2 domain of a polypeptide or a proteindescribed herein is an altered immunoglobulin CH2 region (e.g., analtered human IgG1 CH2 domain) that comprises one or more amino acidsubstitutions at positions 253, 310, 318, 320, 322, and 331. Forexample, an immunoglobulin CH2 region can comprise a substitution atposition 253, 310, 318, 320, 322, or 331, positions 318 and 320,positions 318 and 322, positions 318, 320 and 322, or any othercombination of two, three, four, five or six amino acids at positions253, 310, 318, 320, 322, and 331. In such embodiments, the above-notedmutation(s) decrease or eliminate the CDC activity of a polypeptidecomprising the altered CH2 domain.

In certain other embodiments, in addition to the amino acid substitutionat position 297, an altered CH2 region of a polypeptide or a proteindescribed herein (e.g., an altered human IgG1 CH2 domain) can furthercomprise one or more (e.g., two, three, four, or five) additionalsubstitutions at positions 234-238. For example, an immunoglobulin CH2region can comprise a substitution at positions 234 and 297, positions234, 235, and 297, positions 234, 236 and 297, positions 234-236 and297, positions 234, 235, 237 and 297, positions 234, 236, 238 and 297,positions 234, 235, 237, 238 and 297, positions 236-238 and 297, or anycombination of two, three, four, or five amino acids at positions234-238 in addition to position 297. In addition or alternatively, analtered CH2 region can comprise one or more (e.g., two, three, four orfive) amino acid deletions at positions 234-238, such as at position 236or position 237. The additional mutation(s) decreases or eliminates theADCC activity or Fc receptor-binding capability of a polypeptidecomprising the altered CH2 domain. In certain embodiments, the aminoacid residues at one or more of positions 234-238 have been replacedwith one or more alanine residues. In further embodiments, only one ofthe amino acid residues at positions 234-238 has been deleted while oneor more of the remaining amino acids at positions 234-238 can besubstituted with another amino acid (e.g., alanine or serine).

In certain embodiments, in addition to one or more (e.g., 2, 3, 4, or 5)amino acid substitutions at positions 234-238, a mutated CH2 region of apolypeptide or a protein described herein (e.g., an altered human IgG1CH2 domain) in a fusion protein of the present disclosure can containone or more (e.g., 2, 3, 4, 5, or 6) additional amino acid substitutions(e.g., substituted with alanine) at one or more positions involved incomplement fixation (e.g., at positions I253, H310, E318, K320, K322, orP331). Examples of mutated immunoglobulin CH2 regions include humanIgG1, IgG2, IgG4 and mouse IgG2a CH2 regions with alanine substitutionsat positions 234, 235, 237 (if present), 318, 320 and 322. An exemplarymutated immunoglobulin CH2 region is mouse IGHG2c CH2 region withalanine substitutions at L234, L235, G237, E318, K320, and K322.

In still further embodiments, in addition to the amino acid substitutionat position 297 and the additional deletion(s) or substitution(s) atpositions 234-238, an altered CH2 region of a polypeptide or a proteindescribed herein (e.g., an altered human IgG1 CH2 domain) can furthercomprise one or more (e.g., two, three, four, five, or six) additionalsubstitutions at positions 253, 310, 318, 320, 322, and 331. Forexample, an immunoglobulin CH2 region can comprise a (1) substitution atposition 297, (2) one or more substitutions or deletions or acombination thereof at positions 234-238, and one or more (e.g., 2, 3,4, 5, or 6) amino acid substitutions at positions I253, H310, E318,K320, K322, and P331, such as one, two, three substitutions at positionsE318, K320 and K322. The amino acids at the above-noted positions can besubstituted by alanine or serine.

In certain embodiments, an immunoglobulin CH2 region of a polypeptide ora protein described herein comprises: (i) an amino acid substitution atthe asparagines of position 297 and one amino acid substitution atposition 234, 235, 236 or 237; (ii) an amino acid substitution at theasparagine of position 297 and amino acid substitutions at two ofpositions 234-237; (iii) an amino acid substitution at the asparagine ofposition 297 and amino acid substitutions at three of positions 234-237;(iv) an amino acid substitution at the asparagine of position 297, aminoacid substitutions at positions 234, 235 and 237, and an amino aciddeletion at position 236; (v) amino acid substitutions at three ofpositions 234-237 and amino acid substitutions at positions 318, 320 and322; or (vi) amino acid substitutions at three of positions 234-237, anamino acid deletion at position 236, and amino acid substitutions atpositions 318, 320 and 322.

Exemplary altered immunoglobulin CH2 regions with amino acidsubstitutions at the asparagine of position 297 include: human IgG1 CH2region with alanine substitutions at L234, L235, G237 and N297 and adeletion at G236 (SEQ ID NO: 325 of U.S. Patent Application PublicationNo. 2013/0129723, said sequence incorporated by reference herein), humanIgG2 CH2 region with alanine substitutions at V234, G236, and N297 (SEQID NO: 326 of U.S. Patent Application Publication No. 2013/0129723, saidsequence incorporated by reference herein), human IgG4 CH2 region withalanine substitutions at F234, L235, G237 and N297 and a deletion ofG236 (SEQ ID NO: 322 of U.S. Patent Application Publication No.2013/0129723, said sequence incorporated by reference herein), humanIgG4 CH2 region with alanine substitutions at F234 and N297 (SEQ ID NO:343 of U.S. Patent Application Publication No. US 2013/0129723, saidsequence incorporated by reference herein), human IgG4 CH2 region withalanine substitutions at L235 and N297 (SEQ ID NO: 344 of U.S. PatentApplication Publication No. 2013/0129723, said sequence incorporated byreference herein), human IgG4 CH2 region with alanine substitutions atG236 and N297 (SEQ ID NO: 345 of U.S. Patent Application Publication No.2013/0129723, said sequence incorporated by reference herein), and humanIgG4 CH2 region with alanine substitutions at G237 and N297 (SEQ ID NO:346 of U.S. Patent Application Publication No. 2013/0129723, saidsequence incorporated by reference herein). These CH2 regions can beused in a polypeptide of the present disclosure (e.g., a 5T4-bindingpolypeptide and/or a bispecific 4-1BB polypeptide).

In certain embodiments, in addition to the amino acid substitutionsdescribed above, an altered CH2 region of a polypeptide or a proteindescribed herein (e.g., an altered human IgG1 CH2 domain) can containone or more additional amino acid substitutions at one or more positionsother than the above-noted positions. Such amino acid substitutions canbe conservative or non-conservative amino acid substitutions. Forexample, in certain embodiments, P233 can be changed to E233 in analtered IgG2 CH2 region (see, e.g., SEQ ID NO: 326 of U.S. PatentApplication Publication No. 2013/0129723, said sequence incorporated byreference herein). In addition or alternatively, in certain embodiments,the altered CH2 region can contain one or more amino acid insertions,deletions, or both. The insertion(s), deletion(s) or substitution(s) canbe anywhere in an immunoglobulin CH2 region, such as at the N- orC-terminus of a wild type immunoglobulin CH2 region resulting fromlinking the CH2 region with another region (e.g., a binding domain or animmunoglobulin heterodimerization domain) via a hinge.

In certain embodiments, an altered CH2 domain of a polypeptide orprotein described herein is a human IgG1 CH2 domain with alaninesubstitutions at positions 235, 318, 320, and 322 (i.e., a human IgG1CH2 domain with L235A, E318A, K320A and K322A substitutions) (SEQ ID NO:595 of U.S. Patent Application Publication No. 2013/0129723, saidsequence incorporated by reference herein), and optionally an N297mutation (e.g., to alanine). In certain other embodiments, an alteredCH2 domain is a human IgG1 CH2 domain with alanine substitutions atpositions 234, 235, 237, 318, 320 and 322 (i.e., a human IgG1 CH2 domainwith L234A, L235A, G237A, E318A, K320A and K322A substitutions) (SEQ IDNO: 596 of U.S. Patent Application Publication No. 2013/0129723, saidsequence incorporated by reference herein), and optionally an N297mutation (e.g., to alanine).

In some embodiments, an immunoglobulin constant region of a polypeptideor a protein described herein (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof) comprises a human IgG1 CH2 domain comprising the substitutionsL234A, L235A, G237A, and K322A, according to the EU numbering system.

The CH3 domain that can form an immunoglobulin constant region of apolypeptide or a protein described herein (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) can be a wild type immunoglobulin CH3 domain or analtered immunoglobulin CH3 domain thereof from certain immunoglobulinclasses or subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD,IgE, IgM) of various species (including human, mouse, rat, and othermammals). In certain embodiments, a CH3 domain of a polypeptidedescribed herein (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof) is a wildtype human immunoglobulin CH3 domain, such as wild type CH3 domains ofhuman IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM as set forthin SEQ ID NOs: 116, 208-210, 204-207, and 212, respectively of U.S.Patent Application Publication No. 2013/0129723 (said sequencesincorporated by reference herein). In certain embodiments, the CH3domain is a wild type human IgG1 CH3 domain as set forth in SEQ ID NO:116 of U.S. Patent Application Publication No. 2013/0129723 (saidsequence incorporated by reference herein).

In certain embodiments, a CH3 domain of a polypeptide described herein(e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) is an altered humanimmunoglobulin CH3 domain, such as an altered CH3 domain based on orderived from a wild-type CH3 domain of human IgG1, IgG2, IgG3, IgG4,IgA1, IgA2, IgD, IgE, or IgM antibodies. For example, an altered CH3domain can be a human IgG1 CH3 domain with one or two mutations atpositions H433 and N434 (positions are numbered according to EUnumbering). The mutations in such positions can be involved incomplement fixation. In certain other embodiments, an altered CH3 domainof a polypeptide described herein (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof) can be a human IgG1 CH3 domain but with one or two amino acidsubstitutions at position F405 or Y407. The amino acids at suchpositions are involved in interacting with another CH3 domain. Incertain embodiments, an altered CH3 domain of polypeptide describedherein can be an altered human IgG1 CH3 domain with its last lysinedeleted. The sequence of this altered CH3 domain is set forth in SEQ IDNO: 761 of U.S. Patent Application Publication No. 2013/0129723 (saidsequence incorporated by reference herein).

In certain embodiments, a polypeptide or a protein described herein(e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) comprises a CH3 domain thatcomprises so called “knobs-into-holes” mutations (see, Marvin and Zhu,Acta Pharmacologica Sinica 26:649-58, 2005; Ridgway et al., ProteinEngineering 9:617-21, 1966). More specifically, mutations can beintroduced into each of the CH3 domains of each polypeptide chain sothat the steric complementarity required for CH3/CH3 associationobligates these two CH3 domains to pair with each other. For example, aCH3 domain in one single chain polypeptide of a polypeptide heterodimercan contain a T366W mutation (a “knob” mutation, which substitutes asmall amino acid with a larger one), and a CH3 domain in the othersingle chain polypeptide of the polypeptide heterodimer can contain aY407A mutation (a “hole” mutation, which substitutes a large amino acidwith a smaller one). Other exemplary knobs-into-holes mutations include(1) a T366Y mutation in one CH3 domain and a Y407T in the other CH3domain, and (2) a T366W mutation in one CH3 domain and T366S, L368A andY407V mutations in the other CH3 domain.

The CH4 domain that can form an immunoglobulin constant region apolypeptide or a protein described herein (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) can be a wild type immunoglobulin CH4 domain or analtered immunoglobulin CH4 domain thereof from IgE or IgM molecules. Incertain embodiments, the CH4 domain of a polypeptide described herein isa wild type human immunoglobulin CH4 domain, such as wild type CH4domains of human IgE and IgM molecules as set forth in SEQ ID NOs: 213and 214, respectively, of U.S. Patent Application Publication No.2013/0129723 (said sequences incorporated by reference herein). Incertain embodiments, a CH4 domain of a polypeptide described herein isan altered human immunoglobulin CH4 domain, such as an altered CH4domain based on or derived from a CH4 domain of human IgE or IgMmolecules, which have mutations that increase or decrease animmunological activity known to be associated with an IgE or IgM Fcregion.

In certain embodiments, an immunoglobulin constant region of apolypeptide or a protein described herein (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) comprises a combination of CH2, CH3 or CH4 domains(i.e., more than one constant region domain selected from CH2, CH3 andCH4). For example, the immunoglobulin constant region can comprise CH2and CH3 domains or CH3 and CH4 domains. In certain other embodiments,the immunoglobulin constant region can comprise two CH3 domains and noCH2 or CH4 domains (i.e., only two or more CH3). The multiple constantregion domains that form an immunoglobulin constant region of thepolypeptides described herein can be based on or derived from the sameimmunoglobulin molecule, or the same class or subclass immunoglobulinmolecules. In certain embodiments, the immunoglobulin constant region isan IgG CH2-CH3 (e.g., IgG1 CH2-CH3, IgG2 CH2-CH3, and IgG4 CH2-CH3) andcan be a human (e.g., human IgG1, IgG2, and IgG4) CH2CH3. For example,in certain embodiments, the immunoglobulin constant region of apolypeptide described herein comprises (1) wild type human IgG1 CH2 andCH3 domains, (2) human IgG1 CH2 with N297A substitution (i.e.,CH2(N297A)) and wild type human IgG1 CH3, or (3) human IgG1 CH2(N297A)and an altered human IgG1 CH3 with the last lysine deleted.Alternatively, the multiple constant region domains of a polypeptide ora protein described herein (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof) can be based on or derived from different immunoglobulinmolecules, or different classes or subclasses immunoglobulin molecules.For example, in certain embodiments, an immunoglobulin constant regioncomprises both human IgM CH3 domain and human IgG1 CH3 domain. Themultiple constant region domains that form an immunoglobulin constantregion of a polypeptide described herein can be directly linked togetheror can be linked to each other via one or more (e.g., about 2-about 10)amino acids.

Exemplary immunoglobulin constant regions that can be used in apolypeptide or a protein described herein (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) are set forth in SEQ ID NOs: 305-309, 321, 323, 341,342, and 762 of U.S. Patent Application Publication No. 2013/0129723(said sequences incorporated by reference herein). Further exemplaryimmunoglobulin constant regions that can be used in a polypeptide or aprotein described herein are provided in Table 4 below.

TABLE 4 Exemplary immunoglobulin constant regions SEQ ID NameAmino Acid Sequence NO: SS-FcSSEPKSSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPE 158 domainVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPG DeltaEPKSSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK 160 SS-FcFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIE domainKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPG

In certain embodiments, the immunoglobulin constant regions of eachpolypeptide chain of a homodimeric or heterodimeric protein describedherein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides,and/or multispecific binding proteins thereof) are identical to eachother. In certain other embodiments, the immunoglobulin constant regionof one polypeptide chain of a heterodimeric protein is different fromthe immunoglobulin constant region of the other polypeptide chain of theheterodimer. For example, one immunoglobulin constant region of aheterodimeric protein can contain a CH3 domain with a “knob” mutation,whereas the other immunoglobulin constant region of the heterodimericprotein can contain a CH3 domain with a “hole” mutation.

In some embodiments, the polypeptides of the present disclosure maycomprise an immunoglobulin constant region comprising any of the abovedescribed mutations and a binding domain comprising one or more aminoacid mutations compared to a parental binding domain amino acidsequence. For example, in some embodiments the polypeptides of thepresent disclosure may comprise an immunoglobulin constant regioncomprising one or more of the L234A, L235A, G237A, and K322A mutationsin the human IgG1 CH2 domain and a 5T4-binding domain comprising a V_(H)domain selected from the group consisting of SEQ ID NOs: 38 and 46 and aV_(L) domain selected from the group consisting of SEQ ID NOs: 44, 48,and 50. In some embodiments, the polypeptides of the present disclosuremay comprise an immunoglobulin constant region comprising one or more ofthe L234A, L235A, G237A, and K322A mutations in the human IgG1 CH2domain and a 41BB-binding domain comprising a V_(H) domain amino acidsequence of SEQ ID NO: 14 and a V_(L) domain amino acid sequence of SEQID NO: 16. In some embodiments the polypeptides of the presentdisclosure may comprise an immunoglobulin constant region comprising oneor more of the L234A, L235A, G237A, and K322A mutations in the humanIgG1 CH2 domain and a 5T4-binding domain comprising a V_(H) domainselected from the group consisting of SEQ ID NOs: 38 and 46 and a V_(L)domain selected from the group consisting of SEQ ID NOs: 44, 48, and 50;and a 41BB-binding domain comprising a V_(H) domain amino acid sequenceof SEQ ID NO: 14, and a V_(L) domain amino acid sequence of SEQ ID NO:16.

Polypeptides and proteins described herein (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) may be made using scaffolding as generally disclosedin U.S. Patent Application Publication Nos. 2013/0129723 and2013/0095097, which are each incorporated herein by reference in theirentirety. The polypeptides described herein may comprise twonon-identical polypeptide chains, each polypeptide chain comprising animmunoglobulin heterodimerization domain. The interfacing immunoglobulinheterodimerization domains are different. In one embodiment, theimmunoglobulin heterodimerization domain comprises a CH1 domain or aderivative thereof. In another embodiment, the immunoglobulinheterodimerization domain comprises a CL domain or a derivative thereof.In one embodiment, the CL domain is a Cκ or Cλ isotype or a derivativethereof.

In some embodiments, polypeptides and proteins described herein (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) may have improvedcharacteristics compared to other 5T4-binding polypeptides or4-1BB-binding polypeptides. For example, the 5T4-binding polypeptides,4-1BB-binding polypeptides, or multispecific proteins thereof of thepresent invention may exhibit a reduced isoelectric point compared tothe isoelectric point of a different 5T4-binding polypeptide and/or4-1BB-binding polypeptide. “Isoelectric point” or “pI” is the pH atwhich net charge is zero. The isoelectric point of a protein may bemeasured by any suitable method, e.g., analytical capillary isoelectricfocusing chromatography.

In some embodiments, polypeptides and proteins described herein (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) may bind to 5T4 (e.g., human5T4) and/or 4-1BB with a higher affinity than a previously known 5T4-and/or 4-1BB-binding domains and/or a parent 5T4- and/or 4-1BB-bindingdomain or protein. In some embodiments, the dissociation constant of a5T4- and/or 4-1BB-binding domain or polypeptide may be about 2-5 nM. Incertain embodiments, the off rate of a 5T4- and/or 4-1BB-binding domainor polypeptide may be 4- to 10-fold reduced compared to the off rate ofa previously known antibody or scFv construct or the parent 5T4- and/or4-1BB-binding domain or protein.

In some embodiments, polypeptides and proteins described herein (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) may have a low level of highmolecular weight aggregates produced during recombinant expression ofthe polypeptide or protein. In some embodiments, polypeptides andproteins described herein (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof) may exhibitlonger stability in human serum, depending on the combination of domainspresent in the polypeptide or protein.

5T4-Binding Domains and Proteins Comprising the Same

In some embodiments, the present invention provides 5T4-binding domainsthat specifically bind to 5T4 (e.g. human 5T4). In some embodiments, thepresent invention further provides polypeptides comprising a 5T4-bindingdomain (e.g., 5T4-binding polypeptides). In certain variations, the5T4-binding polypeptide comprises a hinge region carboxyl-terminal tothe 5T4-binding domain, and an immunoglobulin constant region. Infurther variations, the 5T4-binding polypeptide comprises acarboxyl-terminus binding domain linker carboxyl-terminal to theimmunoglobulin constant region, and a second binding domaincarboxyl-terminal to the carboxyl-terminus linker. In yet othervariations, a 5T4-binding polypeptide comprises a hinge regionamino-terminal to the polypeptide comprising the 5T4-binding domain, andan immunoglobulin constant amino-terminal to the hinge region.

In some embodiments, the 5T4-binding domains described herein binds anepitope located on the extracellular domain of 5T4 (e.g., an epitopecomprised within SEQ ID NO: 168). In certain aspects, this epitope is adiscontinuous and/or conformational epitope.

A 5T4-binding domain polypeptide may specifically bind to human 5T4 andcomprise a heavy chain CDR1 (HCDR1), HCDR2, HCDR3, light chain CDR1(LCDR1), LCDR2, and LCDR3, wherein the HCDR1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 30, 52, and60; the HCDR2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 32 and 62; the HCDR3 comprises an amino acidsequence of SEQ ID NO: 34; the LCDR1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 42, and 54; theLCDR2 comprises an amino acid sequence of SEQ ID NOs: 10; and the LCDR3comprises an amino acid sequence of SEQ ID NO: 36.

In particular embodiments, a 5T4-binding domain polypeptide mayspecifically bind to human 5T4 and comprise an HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises an amino acidsequence set forth in SEQ ID NO: 30; (b) the HCDR2 comprises an aminoacid sequence set forth in SEQ ID NO: 32; (c) the HCDR3 comprises anamino acid sequence set forth in SEQ ID NO: 34; (d) the LCDR1 comprisesan amino acid sequence set forth in SEQ ID NO: 8; the LCDR2 comprises anamino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 36. In otherparticular embodiments, a 5T4-binding domain polypeptide specificallybinds to human 5T4 and comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3, wherein (a) the HCDR1 comprises an amino acid sequence setforth in SEQ ID NO: 30; (b) the HCDR2 comprises an amino acid sequenceset forth in SEQ ID NO: 32; (c) the HCDR3 comprises an amino acidsequence set forth in SEQ ID NO: 34; (d) the LCDR1 comprises an aminoacid sequence set forth in SEQ ID NO: 42; (e) the LCDR2 comprises anamino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 36. In otherparticular embodiments, a 5T4-binding domain polypeptide specificallybinds to human 5T4 and comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3, wherein (a) the HCDR1 comprises an amino acid sequence setforth in SEQ ID NO: 52; (b) the HCDR2 comprises an amino acid sequenceset forth in SEQ ID NO: 32; (c) the HCDR3 comprises an amino acidsequence set forth in SEQ ID NO: 34; (d) the LCDR1 comprises an aminoacid sequence set forth in SEQ ID NO: 54; (e) the LCDR2 comprises anamino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 36. In otherparticular embodiments, a 5T4-binding domain polypeptide specificallybinds to human 5T4 and comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3, wherein (a) the HCDR1 comprises an amino acid sequence setforth in SEQ ID NO: 60; (b) the HCDR2 comprises an amino acid sequenceset forth in SEQ ID NO: 62; (c) the HCDR3 comprises an amino acidsequence set forth in SEQ ID NO: 34; (d) the LCDR1 comprises an aminoacid sequence set forth in SEQ ID NO: 54; (e) the LCDR2 comprises anamino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 36.

In certain embodiments, a 5T4-binding domain polypeptide comprises or isa sequence that is at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, at least about 99.5%, or 100% identicalto an amino acid sequence of a light chain variable region (V_(L))selected from the group consisting of SEQ ID NOs: 40, 44, 48, 50, 58,68, and 70, wherein the polypeptide is a multispecific polypeptide inthe format scFv-Fc-scFv and comprises a Y to F substitution in the LCDR1at position 99 of the V_(L) and/or a F to S substitution in the FR3 atposition 148 of the V_(L). In certain embodiments, a polypeptidecomprising a 5T4-binding domain comprises an amino acid sequence of alight chain variable region (V_(L)) selected from the group consistingof SEQ ID NOs: 40, 44, 48, 50, 58, 68, and 70. In certain embodiments, a5T4-binding domain polypeptide comprises an amino acid sequence of aheavy chain variable region (V_(H)) selected from the group consistingof SEQ ID NOs 38, 46, 56, and 64.

In certain embodiments, a 5T4-binding domain polypeptide comprises aV_(H) region comprising the amino acid sequence of SEQ ID NO: 38 and aV_(L) region comprising the amino acid sequence of SEQ ID NO: 40. Incertain embodiments, a 5T4-binding domain polypeptide comprises a V_(H)region comprising the amino acid sequence of SEQ ID NO: 38 and a V_(L)region comprising the amino acid sequence of SEQ ID NO: 44. In certainembodiments, a 5T4-binding domain polypeptide comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 46 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 48. In certainembodiments, a 5T4-binding domain polypeptide comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 46 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 50. In certainembodiments, a 5T4-binding domain polypeptide comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 56 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 58. In certainembodiments, a 5T4-binding domain polypeptide comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 64 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 58. In certainembodiments, a 5T4-binding domain polypeptide comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 38 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 68. In certainembodiments, a 5T4-binding domain polypeptide comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 46 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 70.

Exemplary anti-5T4 binding domain sequences are shown in Table 5.

TABLE 55T4-binding domain polypeptide CDR and variable region amino acid and DNAsequences DNA AA Construct Component DNA Sequence SEQ ID AA sequenceSEQ ID ALG.APV- HCDR1 GGCTTCACATTCAGCAGCTATGCT 29 GFTFSSYA 30 178 HCDR2ATCTCCGGCAGCGGCGGAAGCACC 31 ISGSGGST 32 ALG.APV- HCDR3GCCAGGTACTATGGCGGCTACTAC 33 ARYYGGYYSAWMDY 34 208 TCCGCCTGGATGGACTACLCDR1 CAGTCCATCTCCAGCTAT  7 QSISSY  8 LCDR2 GCCGCTTCC  9 AAS 10 LCDR3CAGCAGACCTATGGCTACCTGCAC 35 QQTYGYLHT 36 ACC V_(H)GAAGTGCAGCTGCTGGAGTCCGGA 37 EVQLLESGGGLVQPGGS 38GGAGGACTGGTGCAGCCTGGCGGA LRLSCAASGFTFSSYAM AGCCTGAGGCTGAGCTGCGCTGCCSWVRQAPGKGLEWVSAI TCCGGCTTCACATTCAGCAGCTAT SGSGGSTYYADSVKGRFGCTATGAGCTGGGTGAGGCAAGCC TISRDNSKNTLYLQMNS CCTGGAAAGGGCCTGGAGTGGGTGLRAEDTAVYYCARYYGG TCCGCTATCTCCGGCAGCGGCGGA YYSAWMDYWGQGTLVTVAGCACCTACTACGCTGACTCCGTC SS AAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGC GCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGA CAGGGCACACTGGTGACCGTGTCC AGC V_(L)GATATTCAGATGACACAGTCCCCT 39 DIQMTQSPSSLSASVGD 40AGCTCCCTGTCCGCCAGCGTGGGA RVTITCRASQSISSYLN GATCGGGTGACCATCACCTGCAGGWYQQKPGKAPKLLIYAA GCCAGCCAGTCCATCTCCAGCTAT SSLQSGVPSRFSGSGSGTTAAACTGGTACCAGCAGAAGCCT TDFTLTISSLQPEDSAT GGAAAGGCTCCCAAGCTGCTGATCYYCQQTYGYLHTFGQGT TACGCCGCTTCCAGCCTCCAGAGC KLEIKGGCGTGCCTAGCAGGTTCTCCGGC TCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCC GAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCAC ACCTTCGGCCAGGGCACAAAGCTG GAGATCAAG ALG.APV-HCDR1 GGCTTCACATTCAGCAGCTATGCT 29 GFTFSSYA 30 179 HCDR2ATCTCCGGCAGCGGCGGAAGCACC 31 ISGSGGST 32 ALG.APV- HCDR3GCCAGGTACTATGGCGGCTACTAC 33 ARYYGGYYSAWMDY 34 209 TCCGCCTGGATGGACTACALG.APV- LCDR1 CAGTCCATCTCCAGCTTC 41 QSISSF 42 222 LCDR2 GCCGCTTCC  9AAS 10 LCDR3 CAGCAGACCTATGGCTACCTGCAC 35 QQTYGYLHT 36 ACC V_(H)GAAGTGCAGCTGCTGGAGTCCGGA 37 EVQLLESGGGLVQPGGS 38GGAGGACTGGTGCAGCCTGGCGGA LRLSCAASGFTFSSYAM AGCCTGAGGCTGAGCTGCGCTGCCSWVRQAPGKGLEWVSAI TCCGGCTTCACATTCAGCAGCTAT SGSGGSTYYADSVKGRFGCTATGAGCTGGGTGAGGCAAGCC TISRDNSKNTLYLQMNS CCTGGAAAGGGCCTGGAGTGGGTGLRAEDTAVYYCARYYGG TCCGCTATCTCCGGCAGCGGCGGA YYSAWMDYWGQGTLVTVAGCACCTACTACGCTGACTCCGTC SS AAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGC GCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGA CAGGGCACACTGGTGACCGTGTCC AGC V_(L)GATATTCAGATGACACAGTCCCCT 43 DIQMTQSPSSLSASVGD 44AGCTCCCTGTCCGCCAGCGTGGGA RVTITCRASQSISSFLN GATCGGGTGACCATCACCTGCAGGWYQQKPGKAPKLLIYAA GCCAGCCAGTCCATCTCCAGCTTC SSLQSGVPSRFSGSGSGTTAAACTGGTACCAGCAGAAGCCT TDFTLTISSLQPEDSAT GGAAAGGCTCCCAAGCTGCTGATCYYCQQTYGYLHTFGQGT TACGCCGCTTCCAGCCTCCAGAGC KLEIKGGCGTGCCTAGCAGGTTCTCCGGC TCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCC GAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCAC ACCTTCGGCCAGGGCACAAAGCTG GAGATCAAG ALG.APV-HCDR1 GGCTTCACATTCAGCAGCTATGCT 29 GFTFSSYA 30 187 HCDR2ATCTCCGGCAGCGGCGGAAGCACC 31 ISGSGGST 32 ALG.APV- HCDR3GCCAGGTACTATGGCGGCTACTAC 33 ARYYGGYYSAWMDY 34 210 TCCGCCTGGATGGACTACALG.APV- LCDR1 CAGTCCATCTCCAGCTTC 41 QSISSF 42 223 LCDR2 GCCGCTTCC  9AAS 10 LCDR3 CAGCAGACCTATGGCTACCTGCAC 35 QQTYGYLHT 36 ACC V_(H)GAAGTGCAGCTGCTGGAGTCCGGA 45 EVQLLESGGGLVQPGGS 46GGAGGACTGGTGCAGCCTGGCGGA LRLSCAASGFTFSSYAM AGCCTGAGGCTGAGCTGCGCTGCCSWVRQAPGKCLEWVSAI TCCGGCTTCACATTCAGCAGCTAT SGSGGSTYYADSVKGRFGCTATGAGCTGGGTGAGGCAAGCC TISRDNSKNTLYLQMNS CCTGGAAAGTGCCTGGAGTGGGTGLRAEDTAVYYCARYYGG TCCGCTATCTCCGGCAGCGGCGGA YYSAWMDYWGQGTLVTVAGCACCTACTACGCTGACTCCGTC SS AAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGC GCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGA CAGGGCACACTGGTGACCGTGTCC AGC V_(L)GATATTCAGATGACACAGTCCCCT 47 DIQMTQSPSSLSASVGD 48AGCTCCCTGTCCGCCAGCGTGGGA RVTITCRASQSISSFLN GATCGGGTGACCATCACCTGCAGGWYQQKPGKAPKLLIYAA GCCAGCCAGTCCATCTCCAGCTTC SSLQSGVPSRFSGSGSGTTAAACTGGTACCAGCAGAAGCCT TDFTLTISSLQPEDSAT GGAAAGGCTCCCAAGCTGCTGATCYYCQQTYGYLHTFGCGT TACGCCGCTTCCAGCCTCCAGAGC KLEIKGGCGTGCCTAGCAGGTTCTCCGGC TCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCC GAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCAC ACCTTCGGCTGCGGCACAAAGCTG GAGATCAAG ALG.APV-HCDR1 GGCTTCACATTCAGCAGCTATGCT 29 GFTFSSYA 30 191 HCDR2ATCTCCGGCAGCGGCGGAAGCACC 31 ISGSGGST 32 HCDR3 GCCAGGTACTATGGCGGCTACTAC33 ARYYGGYYSAWMDY 34 TCCGCCTGGATGGACTAC LCDR1 CAGTCCATCTCCAGCTTC 41QSISSF 42 LCDR2 GCCGCTTCC  9 AAS 10 LCDR3 CAGCAGACCTATGGCTACCTGCAC 35QQTYGYLHT 36 ACC V_(H) GAAGTGCAGCTGCTGGAGTCCGGA 45 EVQLLESGGGLVQPGGS 46GGAGGACTGGTGCAGCCTGGCGGA LRLSCAASGFTFSSYAM AGCCTGAGGCTGAGCTGCGCTGCCSWVRQAPGKCLEWVSAI TCCGGCTTCACATTCAGCAGCTAT SGSGGSTYYADSVKGRFGCTATGAGCTGGGTGAGGCAAGCC TISRDNSKNTLYLQMNS CCTGGAAAGTGCCTGGAGTGGGTGLRAEDTAVYYCARYYGG TCCGCTATCTCCGGCAGCGGCGGA YYSAWMDYWGQGTLVTVAGCACCTACTACGCTGACTCCGTC SS AAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGC GCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGA CAGGGCACACTGGTGACCGTGTCC AGC V_(L)GATATTCAGATGACACAGTCCCCT 49 DIQMTQSPSSLSASVGD 50AGCTCCCTGTCCGCCAGCGTGGGA RVTITCRASQSISSFLN GATCGGGTGACCATCACCTGCAGGWYQQKPGKAPKLLIYAA GCCAGCCAGTCCATCTCCAGCTTC SSLQSGVPSRFSGSGSGTTAAACTGGTACCAGCAGAAGCCT TDFTLTISSLQPEDFAT GGAAAGGCTCCCAAGCTGCTGATCYYCQQTYGYLHTFGCGT TACGCCGCTTCCAGCCTCCAGAGC KLEIKGGCGTGCCTAGCAGGTTCTCCGGC TCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCC GAGGACTTCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCAC ACCTTCGGCTGCGGCACAAAGCTG GAGATCAAG ALG.APV-HCDR1 GGCTTCGACTTCGAGAGCTATGCT 51 GFDFESYA 52 196 HCDR2ATCTCCGGCAGCGGCGGAAGCACC 31 ISGSGGST 32 ALG.APV- HCDR3GCCAGGTACTATGGCGGCTACTAC 33 ARYYGGYYSAWMDY 34 198 TCCGCCTGGATGGACTACLCDR1 CAGTCCATCAGGAGCGCC 53 QSIRSA 54 LCDR2 GCCGCTTCC  9 AAS 10 LCDR3CAGCAGACCTATGGCTACCTGCAC 35 QQTYGYLHT 36 ACC V_(H)GAAGTGCAGCTGCTGGAGTCCGGA 55 EVQLLESGGGLVQPGGS 56GGAGGACTGGTGCAGCCTGGCGGA LRLSCAASGFDFESYAM AGCCTGAGGCTGAGCTGCGCTGCCSWVRQAPGKCLEWVSAI TCCGGCTTCGACTTCGAGAGCTAT SGSGGSTYYADSVKGRFGCTATGAGCTGGGTGAGGCAAGCC TISRDNSKNTLYLQMNS CCTGGAAAGTGCCTGGAGTGGGTGLRAEDTAVYYCARYYGG TCCGCTATCTCCGGCAGCGGCGGA YYSAWMDYWGQGTLVTVAGCACCTACTACGCTGACTCCGTC SS AAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGC GCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGA CAGGGCACACTGGTGACCGTGTCC AGC V_(L)GATATTCAGATGACACAGTCCCCT 57 DIQMTQSPSSLSASVGD 58AGCTCCCTGTCCGCCAGCGTGGGA RVTITCRASQSIRSALN GATCGGGTGACCATCACCTGCAGGWYQQKPGKAPKLLIYAA GCCAGCCAGTCCATCAGGAGCGCC SSLQSGVPSRFSGSGSGCTGAACTGGTACCAGCAGAAGCCT TDFTLTISSLQPEDFAT GGAAAGGCTCCCAAGCTGCTGATCYYCQQTYGYLHTFGCGT TACGCCGCTTCCAGCCTCCAGAGC KLEIKGGCGTGCCTAGCAGGTTCTCCGGC TCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCC GAGGACTTCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCAC ACCTTCGGCTGCGGCACAAAGCTG GAGATCAAG ALG.APV-HCDR1 GGCTTCGACTTCGACAGCTATGCT 59 GFDFDSYA 60 199 HCDR2ATCTCCGGCAGGGGCGGAAGCACC 61 ISGRGGST 62 HCDR3 GCCAGGTACTATGGCGGCTACTAC33 ARYYGGYYSAWMDY 34 TCCGCCTGGATGGACTAC LCDR1 CAGTCCATCAGGAGCGCC 53QSIRSA 54 LCDR2 GCCGCTTCC  9 AAS 10 LCDR3 CAGCAGACCTATGGCTACCTGCAC 35QQTYGYLHT 36 ACC V_(H) GAAGTGCAGCTGCTGGAGTCCGGA 63 EVQLLESGGGLVQPGGS 64GGAGGACTGGTGCAGCCTGGCGGA LRLSCAASGFDFDSYAM AGCCTGAGGCTGAGCTGCGCTGCCSWVRQAPGKCLEWVSAI TCCGGCTTCGACTTCGACAGCTAT SGRGGSTYYADSVKGRFGCTATGAGCTGGGTGAGGCAAGCC TISRDNSKNTLYLQMNS CCTGGAAAGTGCCTGGAGTGGGTGLRAEDTAVYYCARYYGG TCCGCTATCTCCGGCAGGGGCGGA YYSAWMDYWGQGTLVTVAGCACCTACTACGCTGACTCCGTC SS AAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGC GCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGA CAGGGCACACTGGTGACCGTGTCC AGC V_(L)GATATTCAGATGACACAGTCCCCT 57 DIQMTQSPSSLSASVGD 58AGCTCCCTGTCCGCCAGCGTGGGA RVTITCRASQSIRSALN GATCGGGTGACCATCACCTGCAGGWYQQKPGKAPKLLIYAA GCCAGCCAGTCCATCAGGAGCGCC SSLQSGVPSRFSGSGSGCTGAACTGGTACCAGCAGAAGCCT TDFTLTISSLQPEDFAT GGAAAGGCTCCCAAGCTGCTGATCYYCQQTYGYLHTFGCGT TACGCCGCTTCCAGCCTCCAGAGC KLEIKGGCGTGCCTAGCAGGTTCTCCGGC TCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCC GAGGACTTCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCAC ACCTTCGGCTGCGGCACAAAGCTG GAGATCAAG ALG.APV-HCDR1 GGATTCACCTTTAGCAGCTATGCC 29 GFTFSSYA 30 004 HCDR2ATTAGTGGTAGTGGTGGTAGCACA 31 ISGSGGST 32 HCDR3 GCGCGCTACTACGGTGGTTACTAC33 ARYYGGYYSAWMDY 34 TCTGCTTGGATGGACTAT LCDR1 CAGAGCATTAGCAGCTAT  7QSISSY  8 LCDR2 GCTGCATCC  9 AAS 10 LCDR3 CAACAGACTTACGGTTACCTGCAC 35QQTYGYLHT 36 ACT V_(H) GAGGTGCAGCTGCTCGAGAGCGGG 45 EVQLLESGGGLVQPGGS 46GGAGGCTTGGTACAGCCTGGGGGG LRLSCAASGFTFSSYAM TCCCTGCGCCTCTCCTGTGCAGCCSWVRQAPGKCLEWVSAI AGCGGATTCACCTTTAGCAGCTAT SGSGGSTYYADSVKGRFGCCATGAGCTGGGTCCGCCAGGCT TISRDNSKNTLYLQMNS CCAGGGAAGTGTCTGGAGTGGGTCLRAEDTAVYYCARYYGG TCAGCTATTAGTGGTAGTGGTGGT YYSAWMDYWGQGTLVTVAGCACATACTATGCAGACTCCGTG SS AAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGT GCGCGCTACTACGGTGGTTACTACTCTGCTTGGATGGACTATTGGGGC CAGGGAACCCTGGTCACCGTCTCC TCA V_(L)GACATCCAGATGACCCAGTCTCCA 65 DIQMTQSPSSLSASVGD 66TCCTCCCTGAGCGCATCTGTAGGA RVTITCRASQSISSYLN GACCGCGTCACCATCACTTGCCGGWYQQKPGKAPKLLIYAA GCAAGTCAGAGCATTAGCAGCTAT SSLQSGVPSRFSGSGSGTTAAATTGGTATCAGCAGAAACCA TDFTLTISSLQPEDFAT GGGAAAGCCCCTAAGCTCCTGATCYYCQQTYGYLHTFGCGT TATGCTGCATCCAGTTTGCAAAGT RLEIKGGGGTCCCATCACGTTTCAGTGGC AGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT GAAGATTTTGCAACTTATTACTGTCAACAGACTTACGGTTACCTGCAC ACTTTTGGCTGTGGGACCAGGCTG GAGATCAAA ALG.APV-HCDR1 GGATTCACCTTTAGCAGCTATGCC 29 GFTFSSYA 30 006 HCDR2ATTAGTGGTAGTGGTGGTAGCACA 31 ISGSGGST 32 ALG.APV- HCDR3GCGCGCTACTACGGTGGTTACTAC 33 ARYYGGYYSAWMDY 34 010 TCTGCTTGGATGGACTATLCDR1 CAGAGCATTAGCAGCTAT  7 QSISSY  8 LCDR2 GCTGCATCC  9 AAS 10 LCDR3CAACAGACTTACGGTTACCTGCAC 35 QQTYGYLHT 36 ACT V_(H)GAGGTGCAGCTGTTGGAGAGCGGG 37 EVQLLESGGGLVQPGGS 38GGAGGCTTGGTACAGCCTGGGGGG LRLSCAASGFTFSSYAM TCCCTGCGCCTCTCCTGTGCAGCCSWVRQAPGKGLEWVSAI AGCGGATTCACCTTTAGCAGCTAT SGSGGSTYYADSVKGRFGCCATGAGCTGGGTCCGCCAGGCT TISRDNSKNTLYLQMNS CCAGGGAAGGGGCTGGAGTGGGTCLRAEDTAVYYCARYYGG TCAGCTATTAGTGGTAGTGGTGGT YYSAWMDYWGQGTLVTVAGCACATACTATGCAGACTCCGTG SS AAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGT GCGCGCTACTACGGTGGTTACTACTCTGCTTGGATGGACTATTGGGGC CAGGGAACCCTGGTCACCGTCTCC TCA V_(L)GACATCCAGATGACCCAGTCTCCA 67 DIQMTQSPSSLSASVGD 68TCCTCCCTGAGCGCATCTGTAGGA RVTITCRASQSISSYLN GACCGCGTCACCATCACTTGCCGGWYQQKPGKAPKLLIYAA GCAAGTCAGAGCATTAGCAGCTAT SSLQSGVPSRFSGSGSGTTAAATTGGTATCAGCAGAAACCA TDFTLTISSLQPEDFAT GGGAAAGCCCCTAAGCTCCTGATCYYCQQTYGYLHTFGQGT TATGCTGCATCCAGTTTGCAAAGT KLEIKGGGGTCCCATCACGTTTCAGTGGC AGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT GAAGATTTTGCAACTTATTACTGTCAACAGACTTACGGTTACCTGCAC ACTTTTGGCCAGGGGACCAAGCTG GAGATCAAA ALG.APV-HCDR1 GGATTCACCTTTAGCAGCTATGCC 29 GFTFSSYA 30 014 HCDR2ATTAGTGGTAGTGGTGGTAGCACA 31 ISGSGGST 32 ALG.APV- HCDR3GCGCGCTACTACGGTGGTTACTAC 33 ARYYGGYYSAWMDY 34 018 TCTGCTTGGATGGACTATLCDR1 CAGAGCATTAGCAGCTAT  7 QSISSY  8 LCDR2 GCTGCATCC  9 AAS 10 LCDR3CAACAGACTTACGGTTACCTGCAC 35 QQTYGYLHT 36 ACT V_(H)GAGGTGCAGCTGTTGGAGAGCGGG 45 EVQLLESGGGLVQPGGS 46GGAGGCTTGGTACAGCCTGGGGGG LRLSCAASGFTFSSYAM TCCCTGCGCCTCTCCTGTGCAGCCSWVRQAPGKCLEWVSAI AGCGGATTCACCTTTAGCAGCTAT SGSGGSTYYADSVKGRFGCCATGAGCTGGGTCCGCCAGGCT TISRDNSKNTLYLQMNS CCAGGGAAGTGCCTGGAGTGGGTCLRAEDTAVYYCARYYGG TCAGCTATTAGTGGTAGTGGTGGT YYSAWMDYWGQGTLVTVAGCACATACTATGCAGACTCCGTG SS AAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGT GCGCGCTACTACGGTGGTTACTACTCTGCTTGGATGGACTATTGGGGC CAGGGAACCCTGGTCACCGTCTCC TCA V_(L)GACATCCAGATGACCCAGTCTCCA 69 DIQMTQSPSSLSASVGD 70TCCTCCCTGAGCGCATCTGTAGGA RVTITCRASQSISSYLN GACCGCGTCACCATCACTTGCCGGWYQQKPGKAPKLLIYAA GCAAGTCAGAGCATTAGCAGCTAT SSLQSGVPSRFSGSGSGTTAAATTGGTATCAGCAGAAACCA TDFTLTISSLQPEDFAT GGGAAAGCCCCTAAGCTCCTGATCYYCQQTYGYLHTFGCGT TATGCTGCATCCAGTTTGCAAAGT KLEIKGGGGTCCCATCACGTTTCAGTGGC AGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT GAAGATTTTGCAACTTATTACTGTCAACAGACTTACGGTTACCTGCAC ACTTTTGGCTGCGGGACCAAGCTG GAGATCAAA

In some embodiments, the present invention provides for multispecificpolypeptides (e.g., bispecific polypeptides) comprising a 5T4-bindingdomain. In such embodiments, a 5T4-binding protein or polypeptide cancomprise one or more additional binding domains (e.g., second bindingdomain) that bind a target other than 5T4. These other binding domainscan comprise, for example, a particular cytokine or a molecule thattargets the binding domain polypeptide to, for example, a particularcell type, a toxin, an additional cell receptor, or an antibody. Amultispecific 5T4-binding polypeptide or protein may comprise twobinding domains (the domains can be designed to specifically bind thesame or different targets), a hinge region, a linker (e.g., acarboxyl-terminus or an amino-terminus linker), and an immunoglobulinconstant region. A multispecific 5T4-binding protein may be ahomodimeric protein comprising two identical, disulfide-bondedpolypeptides. In some embodiments the 5T4-binding domains may be derivedfrom a monoclonal antibody that binds to 5T4.

In some embodiments of the disclosure, a 5T4-binding polypeptide iscapable of forming a heterodimer with a second polypeptide chain andcomprises a hinge region (a) immediately amino-terminal to animmunoglobulin constant region (e.g., amino-terminal to a CH2 domainwherein the immunoglobulin constant region includes CH2 and CH3 domains,or amino-terminal to a CH3 domain wherein the immunoglobulin sub-regionsincludes CH3 and CH4 domains), (b) interposed between and connecting abinding domain (e.g., scFv) and a immunoglobulin heterodimerizationdomain, (c) interposed between and connecting a immunoglobulinheterodimerization domain and an immunoglobulin constant region (e.g.,wherein the immunoglobulin constant region includes CH2 and CH3 domainsor CH3 and CH4 domains), (d) interposed between and connecting animmunoglobulin constant region and a binding domain, (e) at theamino-terminus of a polypeptide chain, or (f) at the carboxyl-terminusof a polypeptide chain. A polypeptide chain comprising a hinge region asdescribed herein will be capable of associating with a differentpolypeptide chain to form a heterodimeric protein provided herein, andthe heterodimer formed will contain a binding domain that retains itstarget specificity or its specific target binding affinity.

In some embodiments, the 5T4-binding polypeptide provided herein is apolypeptide comprising two scFvs. In some embodiments, the two scFvscomprise 5T4-binding domains. In certain embodiments, the two scFvscomprise identical 5T4-binding domains. In other embodiments, the twoscFvs comprise different 5T4-binding domains. In other embodiments, thepolypeptide comprises an 5T4-binding domain as a first scFv and aneffector cell binding domain as a second scFv. For example, the effectorcell binding domain may be an scFv specific for 4-1BB.

In some embodiments, a 5T4-binding polypeptide provided herein comprisesan anti-5T4 scFv that is at least about 82%, at least about 85%, atleast about 87%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or 100% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134,and 170, wherein the polypeptide is a multispecific polypeptide in theformat scFv-Fc-scFv and comprises a Y to F substitution in the LCDR1 atposition 99 of the anti-5T4 V_(L) and/or a F to S substitution in theFR3 at position 148 of the anti-5T4 V_(L). In some embodiments, a5T4-binding polypeptide provided herein comprises an anti-5T4 scFv thatcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134, and 170. Aminoacid and nucleic acid sequences for exemplary anti-5T4 scFvs areprovided in Table 6 below.

TABLE 6 Amino Acid and DNA Sequences of Exemplary anti-5T4 scFvs DNA AAName DNA Sequence SEQ ID AA Sequence SEQ ID ALG.APV-GAAGTGCAGCTGCTGGAGTCCGGAGGAGGAC 117 EVQLLESGGGLVQPGG 118 178TGGTGCAGCCTGGCGGAAGCCTGAGGCTGAG SLRLSCAASGFTFSSY ALG.APV-CTGCGCTGCCTCCGGCTTCACATTCAGCAGC AMSWVRQAPGKGLEWV 208TATGCTATGAGCTGGGTGAGGCAAGCCCCTG SAISGSGGSTYYADSV anti-5T4GAAAGGGCCTGGAGTGGGTGTCCGCTATCTC KGRFTISRDNSKNTLYCGGCAGCGGCGGAAGCACCTACTACGCTGAC LQMNSLRAEDTAVYYCTCCGTCAAGGGCAGGTTCACCATCAGCCGGG ARYYGGYYSAWMDYWGACAACAGCAAGAACACCCTGTACCTGCAGAT QGTLVTVSSGGGGSGGGAATAGCCTCAGGGCTGAAGACACCGCTGTG GGSGGGGSGGGGSDIQTACTACTGCGCCAGGTACTATGGCGGCTACT MTQSPSSLSASVGDRVACTCCGCCTGGATGGACTACTGGGGACAGGG TITCRASQSISSYLNWCACACTGGTGACCGTGTCCAGCGGCGGAGGC YQQKPGKAPKLLIYAAGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCG SSLQSGVPSRFSGSGSGAAGCGGAGGAGGAGGCTCCGATATTCAGAT GTDFTLTISSLQPEDSGACACAGTCCCCTAGCTCCCTGTCCGCCAGC ATYYCQQTYGYLHTFGGTGGGAGATCGGGTGACCATCACCTGCAGGG QGTKLEIK CCAGCCAGTCCATCTCCAGCTATTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAG CTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGG AAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACT ACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCCAGGGCACAAAGCTGGAGATCAAG ALG.APV- GAAGTGCAGCTGCTGGAGTCCGGAGGAGGAC119 EVQLLESGGGLVQPGG 120 179 TGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGSLRLSCAASGFTFSSY ALG.APV. CTGCGCTGCCTCCGGCTTCACATTCAGCAGCAMSWVRQAPGKGLEWV 209 TATGCTATGAGCTGGGTGAGGCAAGCCCCTG SAISGSGGSTYYADSVALG.APV GAAAGGGCCTGGAGTGGGTGTCCGCTATCTC KGRFTISRDNSKNTLY 222CGGCAGCGGCGGAAGCACCTACTACGCTGAC LQMNSLRAEDTAVYYC anti-5T4TCCGTCAAGGGCAGGTTCACCATCAGCCGGG ARYYGGYYSAWMDYWGACAACAGCAAGAACACCCTGTACCTGCAGAT QGTLVTVSSGGGGSGGGAATAGCCTCAGGGCTGAAGACACCGCTGTG GGSGGGGSGGGGSDIQTACTACTGCGCCAGGTACTATGGCGGCTACT MTQSPSSLSASVGDRVACTCCGCCTGGATGGACTACTGGGGACAGGG TITCRASQSISSFLNWCACACTGGTGACCGTGTCCAGCGGCGGAGGC YQQKPGKAPKLLIYAAGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCG SSLQSGVPSRFSGSGSGAAGCGGAGGAGGAGGCTCCGATATTCAGAT GTDFTLTISSLQPEDSGACACAGTCCCCTAGCTCCCTGTCCGCCAGC ATYYCQQTYGYLHTFGGTGGGAGATCGGGTGACCATCACCTGCAGGG QGTKLEIK CCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAG CTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGG AAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACT ACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCCAGGGCACAAAGCTGGAGATCAAG ALG.APV- GAAGTGCAGCTGCTGGAGTCCGGAGGAGGAC121 EVQLLESGGGLVQPGG 122 187 TGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGSLRLSCAASGFTFSSY ALG.APV. CTGCGCTGCCTCCGGCTTCACATTCAGCAGCAMSWVRQAPGKCLEWV 210 TATGCTATGAGCTGGGTGAGGCAAGCCCCTG SAISGSGGSTYYADSVALG.APV GAAAGTGCCTGGAGTGGGTGTCCGCTATCTC KGRFTISRDNSKNTLY 223CGGCAGCGGCGGAAGCACCTACTACGCTGAC LQMNSLRAEDTAVYYC anti-5T4TCCGTCAAGGGCAGGTTCACCATCAGCCGGG ARYYGGYYSAWMDYWGACAACAGCAAGAACACCCTGTACCTGCAGAT QGTLVTVSSGGGGSGGGAATAGCCTCAGGGCTGAAGACACCGCTGTG GGSGGGGSGGGGSDIQTACTACTGCGCCAGGTACTATGGCGGCTACT MTQSPSSLSASVGDRVACTCCGCCTGGATGGACTACTGGGGACAGGG TITCRASQSISSFLNWCACACTGGTGACCGTGTCCAGCGGCGGAGGC YQQKPGKAPKLLIYAAGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCG SSLQSGVPSRFSGSGSGAAGCGGAGGAGGAGGCTCCGATATTCAGAT GTDFTLTISSLQPEDSGACACAGTCCCCTAGCTCCCTGTCCGCCAGC ATYYCQQTYGYLHTFGGTGGGAGATCGGGTGACCATCACCTGCAGGG CGTKLEIK CCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAG CTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGG AAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACT ACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAG ALG.APV- GAAGTGCAGCTGCTGGAGTCCGGAGGAGGAC123 EVQLLESGGGLVQPGG 124 191 TGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGSLRLSCAASGFTFSSY anti-5T4 CTGCGCTGCCTCCGGCTTCACATTCAGCAGCAMSWVRQAPGKCLEWV TATGCTATGAGCTGGGTGAGGCAAGCCCCTG SAISGSGGSTYYADSVGAAAGTGCCTGGAGTGGGTGTCCGCTATCTC KGRFTISRDNSKNTLYCGGCAGCGGCGGAAGCACCTACTACGCTGAC LQMNSLRAEDTAVYYCTCCGTCAAGGGCAGGTTCACCATCAGCCGGG ARYYGGYYSAWMDYWGACAACAGCAAGAACACCCTGTACCTGCAGAT QGTLVTVSSGGGGSGGGAATAGCCTCAGGGCTGAAGACACCGCTGTG GGSGGGGSGGGGSDIQTACTACTGCGCCAGGTACTATGGCGGCTACT MTQSPSSLSASVGDRVACTCCGCCTGGATGGACTACTGGGGACAGGG TITCRASQSISSFLNWCACACTGGTGACCGTGTCCAGCGGCGGAGGC YQQKPGKAPKLLIYAAGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCG SSLQSGVPSRFSGSGSGAAGCGGAGGAGGAGGCTCCGATATTCAGAT GTDFTLTISSLQPEDFGACACAGTCCCCTAGCTCCCTGTCCGCCAGC ATYYCQQTYGYLHTFGGTGGGAGATCGGGTGACCATCACCTGCAGGG CGTKLEIK CCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAG CTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGG AAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACT ACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAG ALG.APV- GAAGTGCAGCTGCTGGAGTCCGGAGGAGGAC125 EVQLLESGGGLVQPGG 126 196 TGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGSLRLSCAASGFDFESY anti-5T4 CTGCGCTGCCTCCGGCTTCGACTTCGAGAGCAMSWVRQAPGKCLEWV ALG.APV- TATGCTATGAGCTGGGTGAGGCAAGCCCCTGSAISGSGGSTYYADSV 198 GAAAGTGCCTGGAGTGGGTGTCCGCTATCTC KGRFTISRDNSKNTLYanti-5T4 CGGCAGCGGCGGAAGCACCTACTACGCTGAC LQMNSLRAEDTAVYYCTCCGTCAAGGGCAGGTTCACCATCAGCCGGG ARYYGGYYSAWMDYWGACAACAGCAAGAACACCCTGTACCTGCAGAT QGTLVTVSSGGGGSGGGAATAGCCTCAGGGCTGAAGACACCGCTGTG GGSGGGGSGGGGSDIQTACTACTGCGCCAGGTACTATGGCGGCTACT MTQSPSSLSASVGDRVACTCCGCCTGGATGGACTACTGGGGACAGGG TITCRASQSIRSALNWCACACTGGTGACCGTGTCCAGCGGCGGAGGC YQQKPGKAPKLLIYAAGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCG SSLQSGVPSRFSGSGSGAAGCGGAGGAGGAGGCTCCGATATTCAGAT GTDFTLTISSLQPEDFGACACAGTCCCCTAGCTCCCTGTCCGCCAGC ATYYCQQTYGYLHTFGGTGGGAGATCGGGTGACCATCACCTGCAGGG CGTKLEIK CCAGCCAGTCCATCAGGAGCGCCCTGAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAG CTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGG AAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACT ACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAG ALG.APV- GAAGTGCAGCTGCTGGAGTCCGGAGGAGGAC127 EVQLLESGGGLVQPGG 128 199 TGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGSLRLSCAASGFDFDSY anti-5T4 CTGCGCTGCCTCCGGCTTCGACTTCGACAGCAMSWVRQAPGKCLEWV TATGCTATGAGCTGGGTGAGGCAAGCCCCTG SAISGRGGSTYYADSVGAAAGTGCCTGGAGTGGGTGTCCGCTATCTC KGRFTISRDNSKNTLYCGGCAGGGGCGGAAGCACCTACTACGCTGAC LQMNSLRAEDTAVYYCTCCGTCAAGGGCAGGTTCACCATCAGCCGGG ARYYGGYYSAWMDYWGACAACAGCAAGAACACCCTGTACCTGCAGAT QGTLVTVSSGGGGSGGGAATAGCCTCAGGGCTGAAGACACCGCTGTG GGSGGGGSGGGGSDIQTACTACTGCGCCAGGTACTATGGCGGCTACT MTQSPSSLSASVGDRVACTCCGCCTGGATGGACTACTGGGGACAGGG TITCRASQSIRSALNWCACACTGGTGACCGTGTCCAGCGGCGGAGGC YQQKPGKAPKLLIYAAGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCG SSLQSGVPSRFSGSGSGAAGCGGAGGAGGAGGCTCCGATATTCAGAT GTDFTLTISSLQPEDFGACACAGTCCCCTAGCTCCCTGTCCGCCAGC ATYYCQQTYGYLHTFGGTGGGAGATCGGGTGACCATCACCTGCAGGG CGTKLEIK CCAGCCAGTCCATCAGGAGCGCCCTGAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAG CTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGG AAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACT ACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAG ALG.APV- GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCT129 EVQLLESGGGLVQPGG 130 006 TGGTACAGCCTGGGGGGTCCCTGCGCCTCTCSLRLSCAASGFTFSSY anti-5T4 CTGTGCAGCCAGCGGATTCACCTTTAGCAGCAMSWVRQAPGKGLEWV TATGCCATGAGCTGGGTCCGCCAGGCTCCAG SAISGSGGSTYYADSVGGAAGGGGCTGGAGTGGGTCTCAGCTATTAG KGRFTISRDNSKNTLYTGGTAGTGGTGGTAGCACATACTATGCAGAC LQMNSLRAEDTAVYYCTCCGTGAAGGGCCGGTTCACCATCTCCCGTG ARYYGGYYSAWMDYWGACAATTCCAAGAACACGCTGTATCTGCAAAT QGTLVTVSSGGGGSGGGAACAGCCTGCGTGCCGAGGACACGGCTGTA GGSGGGGSGGGGSDIQTATTATTGTGCGCGCTACTACGGTGGTTACT MTQSPSSLSASVGDRVACTCTGCTTGGATGGACTATTGGGGCCAGGG TITCRASQSISSYLNWAACCCTGGTCACCGTCTCCTCAGGCGGTGGA YQQKPGKAPKLLIYAAGGCAGCGGTGGGGGTGGGTCTGGAGGCGGTG SSLQSGVPSRFSGSGSGCAGTGGCGGCGGAGGCTCTGACATCCAGAT GTDFTLTISSLQPEDFGACCCAGTCTCCATCCTCCCTGAGCGCATCT ATYYCQQTYGYLHTFGGTAGGAGACCGCGTCACCATCACTTGCCGGG QGTKLEIK CAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAG CTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGG AAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATT ACTGTCAACAGACTTACGGTTACCTGCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA ALG.APV- GACATCCAGATGACCCAGTCTCCATCCTCCC169 DIQMTQSPSSLSASVG 170 010 TGAGCGCATCTGTAGGAGACCGCGTCACCATDRVTITCRASQSISSY anti-5T4 CACTTGCCGGGCAAGTCAGAGCATTAGCAGCLNWYQQKPGKAPKLLI TATTTAAATTGGTATCAGCAGAAACCAGGGA YAASSLQSGVPSRFSGAAGCCCCTAAGCTCCTGATCTATGCTGCATC SGSGTDFTLTISSLQPCAGTTTGCAAAGTGGGGTCCCATCACGTTTC EDFATYYCQQTYGYLHAGTGGCAGTGGAAGCGGGACAGATTTCACTC TFGQGTKLEIKGGGGSTCACCATCAGCAGTCTGCAACCTGAAGATTT GGGGSGGGGSGGGGSETGCAACTTATTACTGTCAACAGACTTACGGT VQLLESGGGLVQPGGSTACCTGCACACTTTTGGCCAGGGGACCAAGC LRLSCAASGFTFSSYATGGAGATCAAAGGCGGTGGAGGCAGCGGTGG MSWVRQAPGKGLEWVSGGGTGGGTCTGGAGGCGGTGGCAGTGGCGGC AISGSGGSTYYADSVKGGAGGCTCTGAGGTGCAGCTGTTGGAGAGCG GRFTISRDNSKNTLYLGGGGAGGCTTGGTACAGCCTGGGGGGTCCCT QMNSLRAEDTAVYYCAGCGCCTCTCCTGTGCAGCCAGCGGATTCACC RYYGGYYSAWMDYWGQTTTAGCAGCTATGCCATGAGCTGGGTCCGCC GTLVTVSS AGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATAC TATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTA TCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACG GTGGTTACTACTCTGCTTGGATGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA ALG.APV- GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCT131 EVQLLESGGGLVQPGG 132 014 TGGTACAGCCTGGGGGGTCCCTGCGCCTCTCSLRLSCAASGFTFSSY anti-5T4 CTGTGCAGCCAGCGGATTCACCTTTAGCAGCAMSWVRQAPGKCLEWV TATGCCATGAGCTGGGTCCGCCAGGCTCCAG SAISGSGGSTYYADSVGGAAGTGCCTGGAGTGGGTCTCAGCTATTAG KGRFTISRDNSKNTLYTGGTAGTGGTGGTAGCACATACTATGCAGAC LQMNSLRAEDTAVYYCTCCGTGAAGGGCCGGTTCACCATCTCCCGTG ARYYGGYYSAWMDYWGACAATTCCAAGAACACGCTGTATCTGCAAAT QGTLVTVSSGGGGSGGGAACAGCCTGCGTGCCGAGGACACGGCTGTA GGSGGGGSGGGGSDIQTATTATTGTGCGCGCTACTACGGTGGTTACT MTQSPSSLSASVGDRVACTCTGCTTGGATGGACTATTGGGGCCAGGG TITCRASQSISSYLNWAACCCTGGTCACCGTCTCCTCAGGCGGTGGA YQQKPGKAPKLLIYAAGGCAGCGGTGGGGGTGGGTCTGGAGGCGGTG SSLQSGVPSRFSGSGSGCAGTGGCGGCGGAGGCTCTGACATCCAGAT GTDFTLTISSLQPEDFGACCCAGTCTCCATCCTCCCTGAGCGCATCT ATYYCQQTYGYLHTFGGTAGGAGACCGCGTCACCATCACTTGCCGGG CGTKLEIK CAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAG CTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGG AAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATT ACTGTCAACAGACTTACGGTTACCTGCACACTTTTGGCTGCGGGACCAAGCTGGAGATCAAA ALG.APV- GACATCCAGATGACCCAGTCTCCATCCTCCC133 DIQMTQSPSSLSASVG 134 018 TGAGCGCATCTGTAGGAGACCGCGTCACCATDRVTITCRASQSISSY anti-5T4 CACTTGCCGGGCAAGTCAGAGCATTAGCAGCLNWYQQKPGKAPKLLI TATTTAAATTGGTATCAGCAGAAACCAGGGA YAASSLQSGVPSRFSGAAGCCCCTAAGCTCCTGATCTATGCTGCATC SGSGTDFTLTISSLQPCAGTTTGCAAAGTGGGGTCCCATCACGTTTC EDFATYYCQQTYGYLHAGTGGCAGTGGAAGCGGGACAGATTTCACTC TFGCGTKLEIKGGGGSTCACCATCAGCAGTCTGCAACCTGAAGATTT GGGGSGGGGSGGGGSETGCAACTTATTACTGTCAACAGACTTACGGT VQLLESGGGLVQPGGSTACCTGCACACTTTTGGCTGCGGGACCAAGC LRLSCAASGFTFSSYATGGAGATCAAAGGCGGTGGAGGCAGCGGTGG MSWVRQAPGKCLEWVSGGGTGGGTCTGGAGGCGGTGGCAGTGGCGGC AISGSGGSTYYADSVKGGAGGCTCTGAGGTGCAGCTGTTGGAGAGCG GRFTISRDNSKNTLYLGGGGAGGCTTGGTACAGCCTGGGGGGTCCCT QMNSLRAEDTAVYYCAGCGCCTCTCCTGTGCAGCCAGCGGATTCACC RYYGGYYSAWMDYWGQTTTAGCAGCTATGCCATGAGCTGGGTCCGCC GTLVTVSS AGGCTCCAGGGAAGTGCCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATAC TATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTA TCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACG GTGGTTACTACTCTGCTTGGATGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

In some embodiments, a 5T4-binding polypeptide comprises, in order fromamino-terminus to carboxyl-terminus (or in order from carboxyl-terminusto amino-terminus), (i) a 5T4-binding domain, (ii) a hinge region, (iii)an immunoglobulin constant region, (iv) a carboxyl-terminus linker (oran amino-terminus linker), and (v) a second binding domain. In furtherembodiments, the second binding domain is an scFv specific for 4-1BB. Insome embodiments, a 5T4-binding polypeptide comprises, in order fromamino-terminus to carboxyl-terminus (or in order from carboxyl-terminusto amino-terminus), (i) second binding domain, (ii) a hinge region,(iii) an immunoglobulin constant region, (iv) a carboxyl-terminus linker(or an amino-terminus linker), and (v) a 5T4-binding domain. In furtherembodiments, the second binding domain comprises or is a 4-1BB-bindingdomain. In certain embodiments, a 5T4-binding protein can comprise aneffector-cell binding domain for recruitment of effector to target cellsexpressing 5T4. In certain embodiments, the effector-cell binding domainspecifically binds to 4-1BB.

In some embodiments, the second binding domain of a 5T4-bindingpolypeptide described herein is a 4-1BB-binding domain and comprises oneor more of the 4-1BB-binding sequences (e.g., CDRs or variable regions)disclosed in PCT Application Publication No WO 2016/185016; PCTApplication No. PCT/EP2017/059656; Dubrot et al., 2010; Gauttier et al.,2014; Kim et al., 2001; McMillin et al., 2006; Melero et al., 1997;Miller et al., 2002; Sallin et al., 2014; Taraban et al., 2002; Uno etal., 2006; Vinay and Kwon, 2012; Wilcox et al., 2002, each of which isincorporated herein by reference in its entirety.

In some embodiments, the second binding domain specifically binds 4-1BBand comprises an immunoglobulin light chain variable region (V_(L)) andan immunoglobulin heavy chain variable region (V_(H)); wherein the V_(L)comprises an amino acid sequence that is at least about 93% identical,at least about 95% identical, at least about 97% identical, at leastabout 98% identical, at least about 99% identical, or 100% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:16 and 22; and wherein the V_(H) comprises an amino acid sequence thatis at least about 93% identical, at least about 95% identical, at leastabout 97% identical, at least about 98% identical, at least about 99%identical, or 100% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 14, 20, 26, and 28, wherein thepolypeptide is a multispecific polypeptide in the format scFv-Fc-scFvand comprises a Y to H substitution in the HCDR1 at position 150 of theV_(H) and/or an R to H substitution in the FR3 at position 127 of theV_(H). In some embodiments, the second binding domain specifically binds4-1BB and comprises a V_(L) and a V_(H); wherein the V_(L) comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 16and 22; and wherein the V_(H) comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 14, 20, 26, and 28.

In some embodiments, the second binding domain is a 4-1BB-binding domainpolypeptide, wherein the polypeptide is an scFv comprising a sequencethat is at least about 82%, at least about 85%, at least about 87%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 110, 112, 114, and 116. Exemplary anti-4-1BB bindingdomain sequences are shown in Tables 6 and 7 and are discussed furtherbelow.

4-1BB-Binding Domains and Proteins Comprising the Same

In some embodiments, the present invention provides 4-1BB-bindingdomains that specifically bind to 4-1BB (e.g. human 4-1BB). 4-1BB isalso known as CD137 or TNFRSF9 and is a member of the tumor necrosisfactor (TNF) receptor family. 4-1BB is expressed on multiple cell types,including activated subsets of T cell (e.g., CD4+ and CD8+ T cells and Tregulatory cells (Tregs)), natural killer (NK) cells, dendritic cells,monocytes, mast cells, and eosinophils. 4-1BB activation on CD8+ T cellssustains and/or augments cellular activation, while 4-1BB activation onCD4+ T cells can initiate cell activation and, in some instance, lead toactivation-induced cell death. 4-1BB activation on Tregs generallysuppresses Treg function. Several studies have demonstrated induction oftumor immunity through the use of antagonists 4-1BB antibodies.Exemplary human 4-1BB nucleotide and amino acid sequences are providedin SEQ ID NOs: 163, 167 and SEQ ID NOs: 164 and 166, respectively.

In some embodiments, the present invention further provides polypeptidescomprising a 4-1BB-binding domain (e.g., 4-1BB-binding polypeptides). Incertain variations, the 4-1BB-binding polypeptide comprises a hingeregion carboxyl-terminal to the 4-1BB-binding domain, and animmunoglobulin constant region. In further variations, the 4-1BB-bindingpolypeptide comprises a carboxyl-terminus binding domain linkercarboxyl-terminal to the immunoglobulin constant region, and a secondbinding domain carboxyl-terminal to the carboxyl-terminus linker.

In yet other variations, a 4-1BB-binding polypeptide comprises a hingeregion amino-terminal to the polypeptide comprising the 4-1BB-bindingdomain, and an immunoglobulin constant amino-terminal to the hingeregion.

In some embodiments, the 4-1BB-binding domains described herein binds anepitope located on the extracellular domain of 4-1BB (e.g., an epitopecomprised within SEQ ID NO: 166). In certain aspects, this epitope is adiscontinuous and/or conformational epitope.

A 4-1BB-binding domain polypeptide may specifically bind to human 4-1BBand comprise a heavy chain CDR1 (HCDR1), HCDR2, HCDR3, light chain CDR1(LCDR1), LCDR2, and LCDR3, wherein the HCDR1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 2, 18, 24;the HCDR2 comprises an amino acid sequence of SEQ ID NO: 4; the HCDR3comprises an amino acid sequence of SEQ ID NO: 6; the LCDR1 comprises anamino acid sequence of SEQ ID NO: 8; the LCDR2 comprises an amino acidsequence of SEQ ID NO: 10; and the LCDR3 comprises an amino acidsequence of SEQ ID NO: 12, wherein the polypeptide is a multispecificpolypeptide in the format scFv-Fc-scFv and comprises a Y to Hsubstitution in the HCDR1 at position 150 of the anti-4-1BB V_(H) and/oran R to H substitution in the FR3 at position 127 of the anti-4-1BBV_(H).

In particular embodiments, a 4-1BB-binding domain polypeptide mayspecifically bind to human 4-1BB and comprise an HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3, wherein (a) the HCDR1 comprises an amino acidsequence set forth in SEQ ID NO: 2; (b) the HCDR2 comprises an aminoacid sequence set forth in SEQ ID NO: 4; (c) the HCDR3 comprises anamino acid sequence set forth in SEQ ID NO: 6; (d) the LCDR1 comprisesan amino acid sequence set forth in SEQ ID NO: 8; the LCDR2 comprises anamino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 12. In otherparticular embodiments, a 5T4-binding domain polypeptide specificallybinds to human 5T4 and comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3, wherein (a) the HCDR1 comprises an amino acid sequence setforth in SEQ ID NO: 18; (b) the HCDR2 comprises an amino acid sequenceset forth in SEQ ID NO: 4; (c) the HCDR3 comprises an amino acidsequence set forth in SEQ ID NO: 6; (d) the LCDR1 comprises an aminoacid sequence set forth in SEQ ID NO: 8; (e) the LCDR2 comprises anamino acid sequence set forth in SEQ ID NO: 10 and (f) the LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 12. In otherparticular embodiments, a 5T4-binding domain polypeptide specificallybinds to human 5T4 and comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3, wherein (a) the HCDR1 comprises an amino acid sequence setforth in SEQ ID NO: 24; (b) the HCDR2 comprises an amino acid sequenceset forth in SEQ ID NO: 4; (c) the HCDR3 comprises an amino acidsequence set forth in SEQ ID NO: 6; (d) the LCDR1 comprises an aminoacid sequence set forth in SEQ ID NO: 8; (e) the LCDR2 comprises anamino acid sequence set forth in SEQ ID NO: 10; and (f) the LCDR3comprises an amino acid sequence set forth in SEQ ID NO: 12.

In certain embodiments, a 4-1BB-binding domain polypeptide comprises anamino acid sequence of a light chain variable region (V_(L)) selectedfrom the group consisting of SEQ ID NOs: 16 and 22. In certainembodiments, a 4-1BB-binding domain polypeptide comprises an amino acidsequence that is at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, at least about 99.5%, or 100% identical to anamino acid of a heavy chain variable region (V_(H)) selected from thegroup consisting of SEQ ID NOs: 14, 20, 26, and 28, wherein thepolypeptide is a multi specific polypeptide in the format scFv-Fc-scFvand comprises a Y to H substitution in the HCDR1 at position 150 of theanti-4-1BB V_(H) and/or an R to H substitution in the FR3 at position127 of the anti-4-1BB V_(H). In certain embodiments, a 4-1BB-bindingdomain polypeptide comprises an amino acid sequence of a heavy chainvariable region (V_(H)) selected from the group consisting of SEQ IDNOs: 14, 20, 26, and 28.

In certain embodiments, a 4-1BB-binding domain polypeptide comprises aV_(H) region comprising the amino acid sequence of SEQ ID NO: 14 and aV_(L) region comprising the amino acid sequence of SEQ ID NO: 16. Incertain embodiments, a 4-1BB-binding domain polypeptide comprises aV_(H) region comprising the amino acid sequence of SEQ ID NO: 20 and aV_(L) region comprising the amino acid sequence of SEQ ID NO: 22. Incertain embodiments, a 4-1BB-binding domain polypeptide comprises aV_(H) region comprising the amino acid sequence of SEQ ID NO: 26 and aV_(L) region comprising the amino acid sequence of SEQ ID NO: 16. Incertain embodiments, a 4-1BB-binding domain polypeptide comprises aV_(H) region comprising the amino acid sequence of SEQ ID NO: 28 and aV_(L) region comprising the amino acid sequence of SEQ ID NO: 16.

Exemplary anti-4-1BB binding domain sequences are shown in Table 7.

TABLE 7Exemplary 4-1BB binding domain polypeptide and nucleic acid sequencesDNA AA Construct Component DNA Sequence SEQ ID AA sequence SEQ IDALG.APV-178 HCDR1 GGATTCACCTTTTCTCACGGTTCT  1 GFTFSHGS  2 ALG.APV-179HCDR2 ATTTCTTCTGGTTCTGGTTCTACA  3 ISSGSGST  4 ALG.APV-187 HCDR3GCGCGCTCTTCTTACTACGGTTCT  5 ARSSYYGSYYSIDY  6 ALG.APV-198TACTACTCTATTGACTAT ALG.APV-199 LCDR1 CAGAGCATTAGCAGCTAT  7 QSISSY  8ALG.APV-208 LCDR2 GCTGCATCC  9 AAS 10 ALG.APV-209 LCDR3CAACAGTACTACGACAACCTGCCC 11 QQYYDNLPT 12 ALG.APV-210 ACT ALG.APV-222V_(H) GAGGTGCAGCTGTTGGAGAGCGGG 13 EVQLLESGGGLVQP 14 ALG.APV-223GGAGGCTTGGTACAGCCTGGGGGG GGSLRLSCAASGFT TCCCTGCGCCTCTCCTGTGCAGCCFSHGSMYWVRQAPG AGCGGATTCACCTTTTCTCACGGT KGLEWVSSISSGSGTCTATGTACTGGGTCCGCCAGGCT STYYADSVKGRFTI CCAGGGAAGGGGCTGGAGTGGGTCSHDNSKNTLYLQMN TCATCTATTTCTTCTGGTTCTGGT SLRAEDTAVYYCARTCTACATACTATGCAGACTCCGTG SSYYGSYYSIDYWG AAGGGCCGGTTCACCATCTCCCATQGTLVTVSS GACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGT GCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGC CAGGGAACCCTGGTCACCGTCTCC TCA V_(L)GACATCCAGATGACCCAGTCTCCA 15 DIQMTQSPSSLSAS 16 TCCTCCCTGAGCGCATCTGTAGGAVGDRVTITCRASQS GACCGCGTCACCATCACTTGCCGG ISSYLNWYQQKPGKGCAAGTCAGAGCATTAGCAGCTAT APKLLIYAASSLQS TTAAATTGGTATCAGCAGAAACCAGVPSRFSGSGSGTD GGGAAAGCCCCTAAGCTCCTGATC FTLTISSLQPEDFATATGCTGCATCCAGTTTGCAAAGT TYYCQQYYDNLPTF GGGGTCCCATCACGTTTCAGTGGCGQGTKLEIK AGTGGAAGCGGGACAGATTTCACT CTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGT CAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTG GAGATCAAA ALG.APV-191 HCDR1GGATTCACCTTTGATTACGGTTCT 17 GFTFDYGS 18 HCDR2 ATTTCTTCTGGTTCTGGTTCTACA 3 ISSGSGST  4 HCDR3 GCGCGCTCTTCTTACTACGGTTCT  5 ARSSYYGSYYSIDY  6TACTACTCTATTGACTAT LCDR1 CAGAGCATTAGCAGCTAT  7 QSISSY  8 LCDR2 GCTGCATCC 9 AAS 10 LCDR3 CAACAGTACTACGACAACCTGCCC 11 QQYYDNLPT 12 ACT V_(H)GAGGTGCAGCTGTTGGAGAGCGGG 19 EVQLLESGGGLVQP 20 GGAGGCTTGGTACAGCCTGGGGGGGGSLRLSCAASGFT TCCCTGCGCCTCTCCTGTGCAGCC FDYGSMYWVRQAPGAGCGGATTCACCTTTGATTACGGT KGLEWVSSISSGSG TCTATGTACTGGGTCCGCCAGGCTSTYYADSVKGRFTI CCAGGGAAGGGGCTGGAGTGGGTC SHDNSKNTLYLQMNTCATCTATTTCTTCTGGTTCTGGT SLRAEDTAVYYCAR TCTACATACTATGCAGACTCCGTGSSYYGSYYSIDYWG AAGGGCCGGTTCACCATCTCCCAC QGTLVTVSSGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGT GCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGC CAGGGAACCCTGGTCACCGTCTCC TCA V_(L)GACATCCAGATGACCCAGTCTCCA 21 DIQMTQSPSSLSAS 22 TCCTCCCTGAGCGCATCTGTAGGAVGDRVTITCRASQS GACCGCGTCACCATCACTTGCCGG ISSYLNWYQQKPGKGCAAGTCAGAGCATTAGCAGCTAT APKLLIYAASSLHS TTAAATTGGTATCAGCAGAAACCAGVPSRFSGSGSGTD GGGAAAGCCCCTAAGCTCCTGATC FTLTISSLQPEDFATATGCTGCATCCAGTTTGCACAGT TYYCQQYYDNLPTF GGGGTCCCATCACGTTTCAGTGGCGQGTKLEIK AGTGGAAGCGGGACAGATTTCACT CTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGT CAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTG GAGATCAAA ALG.APV-196 HCDR1GGATTCACCTTTTCTTACGGTTCT 23 GFTFSYGS 24 HCDR2 ATTTCTTCTGGTTCTGGTTCTACA 3 ISSGSGST  4 HCDR3 GCGCGCTCTTCTTACTACGGTTCT  5 ARSSYYGSYYSIDY  6TACTACTCTATTGACTAT LCDR1 CAGAGCATTAGCAGCTAT  7 QSISSY  8 LCDR2 GCTGCATCC 9 AAS 10 LCDR3 CAACAGTACTACGACAACCTGCCC 11 QQYYDNLPT 12 ACT V_(H)GAGGTGCAGCTGTTGGAGAGCGGG 25 EVQLLESGGGLVQP 26 GGAGGCTTGGTACAGCCTGGGGGGGGSLRLSCAASGFT TCCCTGCGCCTCTCCTGTGCAGCC FSYGSMYWVRQAPGAGCGGATTCACCTTTTCTTACGGT KGLEWVSSISSGSG TCTATGTACTGGGTCCGCCAGGCTSTYYADSVKGRFTI CCAGGGAAGGGGCTGGAGTGGGTC SHDNSKNTLYLQMNTCATCTATTTCTTCTGGTTCTGGT SLRAEDTAVYYCAR TCTACATACTATGCAGACTCCGTGSSYYGSYYSIDYWG AAGGGCCGGTTCACCATCTCCCAT QGTLVTVSSGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGT GCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGC CAGGGAACCCTGGTCACCGTCTCC TCA V_(L)GACATCCAGATGACCCAGTCTCCA 15 DIQMTQSPSSLSAS 16 TCCTCCCTGAGCGCATCTGTAGGAVGDRVTITCRASQS GACCGCGTCACCATCACTTGCCGG ISSYLNWYQQKPGKGCAAGTCAGAGCATTAGCAGCTAT APKLLIYAASSLQS TTAAATTGGTATCAGCAGAAACCAGVPSRFSGSGSGTD GGGAAAGCCCCTAAGCTCCTGATC FTLTISSLQPEDFATATGCTGCATCCAGTTTGCAAAGT TYYCQQYYDNLPTF GGGGTCCCATCACGTTTCAGTGGCGQGTKLEIK AGTGGAAGCGGGACAGATTTCACT CTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGT CAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTG GAGATCAAA ALG.APV-004 HCDR1GGATTCACCTTTTCTTACGGTTCT 23 GFTFSYGS 24 ALG.APV-006 HCDR2ATTTCTTCTGGTTCTGGTTCTACA  3 ISSGSGST  4 ALG.APV-010 HCDR3GCGCGCTCTTCTTACTACGGTTCT  5 ARSSYYGSYYSIDY  6 ALG.APV-014TACTACTCTATTGACTAT ALG.APV-018 LCDR1 CAGAGCATTAGCAGCTAT  7 QSISSY  8LCDR2 GCTGCATCC  9 AAS 10 LCDR3 CAACAGTACTACGACAACCTGCCC 11 QQYYDNLPT 12ACT V_(H) GAGGTGCAGCTGTTGGAGAGCGGG 27 EVQLLESGGGLVQP 28GGAGGCTTGGTACAGCCTGGGGGG GGSLRLSCAASGFT TCCCTGCGCCTCTCCTGTGCAGCCFSYGSMYWVRQAPG AGCGGATTCACCTTTTCTTACGGT KGLEWVSSISSGSGTCTATGTACTGGGTCCGCCAGGCT STYYADSVKGRFTI CCAGGGAAGGGGCTGGAGTGGGTCSRDNSKNTLYLQMN TCATCTATTTCTTCTGGTTCTGGT SLRAEDTAVYYCARTCTACATACTATGCAGACTCCGTG SSYYGSYYSIDYWG AAGGGCCGGTTCACCATCTCCCGTQGTLVTVSS GACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGT GCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGC CAGGGAACCCTGGTCACCGTCTCC TCA V_(L)GACATCCAGATGACCCAGTCTCCA 15 DIQMTQSPSSLSAS 16 TCCTCCCTGAGCGCATCTGTAGGAVGDRVTITCRASQS GACCGCGTCACCATCACTTGCCGG ISSYLNWYQQKPGKGCAAGTCAGAGCATTAGCAGCTAT APKLLIYAASSLQS TTAAATTGGTATCAGCAGAAACCAGVPSRFSGSGSGTD GGGAAAGCCCCTAAGCTCCTGATC FTLTISSLQPEDFATATGCTGCATCCAGTTTGCAAAGT TYYCQQYYDNLPTF GGGGTCCCATCACGTTTCAGTGGCGQGTKLEIK AGTGGAAGCGGGACAGATTTCACT CTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGT CAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTG GAGATCAAA

In some embodiments, the present invention provides for multispecificpolypeptides (e.g., bispecific polypeptides) comprising a 4-1BB-bindingdomain. A multispecific 4-1BB-binding polypeptide or protein maycomprise two binding domains (the domains can be designed tospecifically bind the same or different targets), a hinge region, alinker (e.g., a carboxyl-terminus or an amino-terminus linker), and animmunoglobulin constant region. A multispecific 4-1BB-binding proteinmay be a homodimeric protein comprising two identical, disulfide-bondedpolypeptides. In some embodiments the 4-1BB binding domains may bederived from a monoclonal antibody that binds to 4-1BB (e.g., Urelumab(BMS-66513) or PF-05082566 (Pfizer)), derived from a 4-1BB-bindingdomain described in PCT Application Publication No. WO 2016/185016, orderived from a 4-1BB-binding domain PCT Application No.PCT/EP2017/059656.

In some embodiments, a multispecific polypeptide comprising a 4-1BBbinding domain may comprise, in order from amino-terminus tocarboxyl-terminus: (i) a first binding domain; (ii) a hinge region;(iii) an immunoglobulin constant region; (iv) a binding domain linker;and (v) a 4-1BB-binding domain. In some embodiments, a multispecificpolypeptide comprising a 4-1BB binding domain may comprise, in orderfrom amino-terminus to carboxyl-terminus: (i) a 4-1BB-binding domain;(ii) a binding domain linker; (iii) an immunoglobulin constant region;(iv) a hinge region; and (v) a first binding domain. Amino acid andnucleic acid sequences for exemplary anti-4-1BB binding domain sequencesare provided in Table 7.

In certain embodiments, a 4-1BB-binding protein or polypeptide cancomprise one or more additional binding domains (e.g., second bindingdomain) that bind a target other than 4-1BB. These other binding domainscan comprise, for example, a particular cytokine or a molecule thattargets the binding domain polypeptide to, for example, a particularcell type, a toxin, an additional cell receptor, or an antibody.

In some embodiments of the disclosure, a 4-1BB-binding polypeptide iscapable of forming a heterodimer with a second polypeptide chain andcomprises a hinge region (a) immediately amino-terminal to animmunoglobulin constant region (e.g., amino-terminal to a CH2 domainwherein the immunoglobulin constant region includes CH2 and CH3 domains,or amino-terminal to a CH3 domain wherein the immunoglobulin sub-regionsincludes CH3 and CH4 domains), (b) interposed between and connecting abinding domain (e.g., scFv) and a immunoglobulin heterodimerizationdomain, (c) interposed between and connecting a immunoglobulinheterodimerization domain and an immunoglobulin constant region (e.g.,wherein the immunoglobulin constant region includes CH2 and CH3 domainsor CH3 and CH4 domains), (d) interposed between and connecting animmunoglobulin constant region and a binding domain, (e) at theamino-terminus of a polypeptide chain, or (f) at the carboxyl-terminusof a polypeptide chain. A polypeptide chain comprising a hinge region asdescribed herein will be capable of associating with a differentpolypeptide chain to form a heterodimeric protein provided herein, andthe heterodimer formed will contain a binding domain that retains itstarget specificity or its specific target binding affinity.

In certain embodiments, a 4-1BB-binding polypeptide can comprise atarget cell binding domain for recruitment of target cells to effectorcells expressing 4-1BB. In certain embodiments, the target cell bindingdomain specifically binds to 5T4. In certain embodiments, a4-1BB-binding protein as described herein can comprise (i) a bindingdomain that specifically binds 4-1BB and (ii) another binding domainthat specifically binds to 5T4. Non-limiting examples of anti-5T4antibodies from which the 5T4 binding domain can be derived includethose described in PCT Application Publication No. WO 2016/185016 andPCT Application No. PCT/EP2017/059656.

In some embodiments, the 4-1BB-binding polypeptide provided herein is apolypeptide comprising two scFvs. In some embodiments, the two scFvscomprise 4-1BB-binding domains. In certain embodiments, the two scFvscomprise identical 4-1BB-binding domains. In other embodiments, the twoscFvs comprise different 4-1BB-binding domains. In other embodiments,the polypeptide comprises a 4-1BB-binding domain as a first scFv and antarget cell binding domain as a second scFv. For example, the targetcell binding domain may be an scFv specific for 5T4.

In some embodiments, the bispecific 4-1BB-binding polypeptides andproteins provided herein comprise an anti-4-1BB scFv that is at leastabout 82%, at least about 85%, at least about 87%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or 100% identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 110,112, 114, and 116, wherein the polypeptide is a multispecificpolypeptide in the format scFv-Fc-scFv and comprises a Y to Hsubstitution in the HCDR1 at position 150 of the anti-4-1BB V_(H) and/oran R to H substitution in the FR3 at position 127 of the anti-4-1BBV_(H). In certain embodiments, the bispecific 4-1BB-binding polypeptidesand proteins provided herein comprise an anti-4-1BB scFv that comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:110, 112, 114, and 116. Amino acid and nucleic acid sequences forexemplary anti-4-1BB scFvs are provided in Table 8 below.

TABLE 8 DNA and AA Sequences of Exemplary anti-4-1BB scFvs DNA AAConstruct DNA Sequence SEQ ID AA Sequence SEQ ID ALG.APV-178-4-1BBGAGGTGCAGCTGTTGGAGAGCGGGGGAGG 109 EVQLLESGGGLVQPGGSLRL 110ALG.APV-179-4-1BB CTTGGTACAGCCTGGGGGGTCCCTGCGCC SCAASGFTFSHGSMYWVRQAALG.APV-187-4-1BB TCTCCTGTGCAGCCAGCGGATTCACCTTT PGKGLEWVSSISSGSGSTYYALG.APV-198-4-1BB TCTCACGGTTCTATGTACTGGGTCCGCCA ADSVKGRFTISHDNSKNTLYALG.APV-199-4-1BB GGCTCCAGGGAAGGGGCTGGAGTGGGTCT LQMNSLRAEDTAVYYCARSSALG.APV-208-4-1BB CATCTATTTCTTCTGGTTCTGGTTCTACA YYGSYYSIDYWGQGTLVTVSALG.APV-209-4-1BB TACTATGCAGACTCCGTGAAGGGCCGGTT SGGGGSGGGGSGGGGSGGGGALV.APV-210-4-1BB CACCATCTCCCATGACAATTCCAAGAACA SDIQMTQSPSSLSASVGDRVALG.APV-222-4-1BB CGCTGTATCTGCAAATGAACAGCCTGCGT TITCRASQSISSYLNWYQQKALG.APV-223-4-1BB GCCGAGGACACGGCTGTATATTATTGTGC PGKAPKLLIYAASSLQSGVPGCGCTCTTCTTACTACGGTTCTTACTACT SRFSGSGSGTDFTLTISSLQCTATTGACTATTGGGGCCAGGGAACCCTG PEDFATYYCQQYYDNLPTFGGTCACCGTCTCCTCAGGTGGAGGTGGCTC QGTKLEIK CGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATG ACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCC GGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGC CCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTC AGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAG ATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGG GACCAAGCTGGAGATCAAA ALG.APV-191-4-1BBGAGGTGCAGCTGTTGGAGAGCGGGGGAGG 111 EVQLLESGGGLVQPGGSLRL 112CTTGGTACAGCCTGGGGGGTCCCTGCGCC SCAASGFTFDYGSMYWVRQATCTCCTGTGCAGCCAGCGGATTCACCTTT PGKGLEWVSSISSGSGSTYYGATTACGGTTCTATGTACTGGGTCCGCCA ADSVKGRFTISHDNSKNTLYGGCTCCAGGGAAGGGGCTGGAGTGGGTCT LQMNSLRAEDTAVYYCARSSCATCTATTTCTTCTGGTTCTGGTTCTACA YYGSYYSIDYWGQGTLVTVSTACTATGCAGACTCCGTGAAGGGCCGGTT SGGGGSGGGGSGGGGSGGGGCACCATCTCCCACGACAATTCCAAGAACA SDIQMTQSPSSLSASVGDRVCGCTGTATCTGCAAATGAACAGCCTGCGT TITCRASQSISSYLNWYQQKGCCGAGGACACGGCTGTATATTATTGTGC PGKAPKLLIYAASSLHSGVPGCGCTCTTCTTACTACGGTTCTTACTACT SRFSGSGSGTDFTLTISSLQCTATTGACTATTGGGGCCAGGGAACCCTG PEDFATYYCQQYYDNLPTFGGTCACCGTCTCCTCAGGTGGAGGTGGCTC QGTKLEIK CGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATG ACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCC GGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGC CCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCACAGTGGGGTCCCATCACGTTTC AGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAG ATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGG GACCAAGCTGGAGATCAAA ALG.APV-196-4-1BBGAGGTGCAGCTGTTGGAGAGCGGGGGAGG 113 EVQLLESGGGLVQPGGSLRL 114CTTGGTACAGCCTGGGGGGTCCCTGCGCC SCAASGFTFSYGSMYWVRQATCTCCTGTGCAGCCAGCGGATTCACCTTT PGKGLEWVSSISSGSGSTYYTCTTACGGTTCTATGTACTGGGTCCGCCA ADSVKGRFTISHDNSKNTLYGGCTCCAGGGAAGGGGCTGGAGTGGGTCT LQMNSLRAEDTAVYYCARSSCATCTATTTCTTCTGGTTCTGGTTCTACA YYGSYYSIDYWGQGTLVTVSTACTATGCAGACTCCGTGAAGGGCCGGTT SGGGGSGGGGSGGGGSGGGGCACCATCTCCCATGACAATTCCAAGAACA SDIQMTQSPSSLSASVGDRVCGCTGTATCTGCAAATGAACAGCCTGCGT TITCRASQSISSYLNWYQQKGCCGAGGACACGGCTGTATATTATTGTGC PGKAPKLLIYAASSLQSGVPGCGCTCTTCTTACTACGGTTCTTACTACT SRFSGSGSGTDFTLTISSLQCTATTGACTATTGGGGCCAGGGAACCCTG PEDFATYYCQQYYDNLPTFGGTCACCGTCTCCTCAGGTGGAGGTGGCTC QGTKLEIK CGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATG ACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCC GGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGC CCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTC AGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAG ATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGG GACCAAGCTGGAGATCAAA ALG.APV-006-4-1BBGAGGTGCAGCTGTTGGAGAGCGGGGGAGG 115 EVQLLESGGGLVQPGGSLRL 116ALG.APV-010-4-1BB CTTGGTACAGCCTGGGGGGTCCCTGCGCC SCAASGFTFSYGSMYWVRQAALG.APV-014-4-1BB TCTCCTGTGCAGCCAGCGGATTCACCTTT PGKGLEWVSSISSGSGSTYYALG.APV-018-4-1BB TCTTACGGTTCTATGTACTGGGTCCGCCA ADSVKGRFTISRDNSKNTLYGGCTCCAGGGAAGGGGCTGGAGTGGGTCT LQMNSLRAEDTAVYYCARSSCATCTATTTCTTCTGGTTCTGGTTCTACA YYGSYYSIDYWGQGTLVTVSTACTATGCAGACTCCGTGAAGGGCCGGTT SGGGGSGGGGSGGGGSGGGGCACCATCTCCCGTGACAATTCCAAGAACA SDIQMTQSPSSLSASVGDRVCGCTGTATCTGCAAATGAACAGCCTGCGT TITCRASQSISSYLNWYQQKGCCGAGGACACGGCTGTATATTATTGTGC PGKAPKLLIYAASSLQSGVPGCGCTCTTCTTACTACGGTTCTTACTACT SRFSGSGSGTDFTLTISSLQCTATTGACTATTGGGGCCAGGGAACCCTG PEDFATYYCQQYYDNLPTFGGTCACCGTCTCCTCAGGCGGCGGCGGCAG QGTKLEIK CGGCGGCGGCGGCAGCGGCGGCGGAGGCTCCGGCGGCGGCGGCAGCGACATCCAGATG ACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCC GGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGC CCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTC AGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAG ATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGG GACCAAGCTGGAGATCAAA

Bispecific Molecules

In some embodiments, the present invention provides multispecific5T4-binding polypeptides comprising two binding domains wherein onebinding domain is specific for 5T4 and the second binding domain isspecific for a different target antigen. In some embodiments, thepresent invention provides bispecific 4-1BB-binding polypeptidescomprising two binding domains wherein binding domain is specific for4-1BB and the second binding domain is specific for a different targetantigen. In particular embodiments, the present invention providesbispecific polypeptides comprising two binding domains, wherein onebinding domain is specific for 4-1BB and wherein the other bindingdomain is specific for 5T4. Such embodiments are referred to herein asanti-5T4×anti-4-1BB molecules or anti-5T4×anti-4-1BB polypeptides orproteins. In particular embodiments, the 5T4 and 4-1BB binding domainsof an anti-5T4×anti-4-1BB molecule are scFv domains that specificallybind to 5T4 and 4-1BB, respectively.

In some embodiments, the anti-5T4×anti-4-1BB molecules described hereincomprise in order from amino-terminus to carboxyl-terminus (i) a5T4-binding domain; (ii) a binding domain linker; and (iii) a4-1BB-binding domain. In some embodiments, the anti-5T4×anti-4-1BBmolecules described herein comprise in order from amino-terminus tocarboxyl-terminus (i) a 4-1BB-binding domain; (ii) a binding domainlinker; and (iii) a 5T4-binding domain. In some embodiments, theanti-5T4×anti-4-1BB molecules described herein comprise in order fromamino-terminus to carboxyl-terminus (or in order from carboxyl-terminusto amino-terminus), (i) a 5T4-binding domain, (ii) a hinge region, (iii)an immunoglobulin constant region, (iv) a carboxyl-terminus linker (oran amino-terminus linker), and (v) a second binding domain.

The anti-5T4×anti-4-1BB molecules described herein may comprise anycombination of the anti-5T4 and the anti-4-1BB binding domain sequencesshown in Tables 4-7. In particular embodiments, the anti-5T4×anti-4-1BBmolecules of the present invention comprise a 5T4-binding domain and a4-1BB-binding domain that each comprise a HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3. In some embodiments, the anti-5T4×anti-4-1BB moleculescomprise (a) a first scFv domain comprising: (i) V_(H) comprising anHCDR1 amino acid sequence selected from the group consisting of SEQ IDNOs: 30, 52, and 60, an HCDR2 amino acid sequence selected from thegroup consisting of SEQ ID NOs: 32 and 62, and an HCDR3 amino acidsequence of SEQ ID NO: 34; and (ii) a V_(L) comprising an LCDR1 aminoacid sequence selected from the group consisting of SEQ ID NOs: 8, 42,and 54, an LCDR2 amino acid sequence of SEQ ID NO: 10, and an LCDR3amino acid sequence of SEQ ID NO: 36; and (b) a second scFv domaincomprising (i) a V_(H) comprising an HCDR1 amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 2, 18, and 24, an HCDR2 aminoacid sequence of SEQ ID NO: 4, and an HCDR3 amino acid sequence of SEQID NO: 6; and (ii) a V_(L) comprising an LCDR1 amino acid sequence ofSEQ ID NO: 8, an LCDR2 amino acid sequence of SEQ ID NO: 10, and anLCDR3 amino acid sequence of SEQ ID NO: 12.

In some embodiments, the anti-5T4×anti-4-1BB molecules of the presentinvention comprise a 5T4-binding domain and a 4-1BB-binding domain thateach comprise a V_(H) and a V_(L) domain. In some embodiments, theanti-5T4×anti-4-1BB molecules comprise a first scFv domain comprising i)a V_(H) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 38, 46, 56, and 64 and a V_(L) comprising anamino acid selected from the group consisting of SEQ ID NOs: 40, 44, 48,50 58, 66, 68, and 70; and a second scFv domain comprising ii) a V_(H)comprising an amino acid selected from the group consisting of SEQ IDNO: 14, 20, 26, and 28 and a V_(L) comprising an amino acid selectedfrom the group consisting of SEQ ID NO: 16 and 22.

In certain embodiments, the 5T4-binding domain of an anti-5T4×anti-4-1BBmolecule comprises or is an scFv that is at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, at least about 99.5%, or 100% identicalto an amino acid sequence selected from the group consisting of SEQ IDNOs: 118, 120, 122, 124, 126, 128, 130, 132, 134, and 170, wherein themolecule is a multispecific polypeptide in the format scFv-Fc-scFv andcomprises a Y to F substitution in the LCDR1 at position 99 of theanti-5T4 V_(L) and/or a F to S substitution in the FR3 at position 148of the anti-5T4 V_(L). In certain embodiments, the 5T4-binding domain ofan anti-5T4×anti-4-1BB molecule comprises an scFv that comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:118, 120, 122, 124, 126, 128, 130, 132, 134, and 170.

In certain embodiments, the 4-1BB-binding domain of ananti-5T4×anti-4-1BB molecule comprises or is an scFv that is at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, at least about 99.5%,or 100% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 110, 112, 114, and 116, wherein the moleculeis a multispecific polypeptide in the format scFv-Fc-scFv and comprisesa Y to H substitution in the HCDR1 at position 150 of the anti-4-1BBV_(H) and/or an R to H substitution in the FR3 at position 127 of theanti-4-1BB V_(H). In certain embodiments, the 4-1BB-binding domain of ananti-5T4×anti-4-1BB molecule comprises scFv that comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 110, 112,114, and 116.

In particular embodiments, the anti-5T4×anti-4-1BB molecule comprises acombination of CDR sequences, V_(H)/V_(L) sequences, and/or scFvsequences as shown in Table 9 below.

TABLE 9 Exemplary anti-5T4 and anti-4- 1BB Binding Sequence Combinations4-1BB Binding Domain 5T4 Binding Domain Component SEQ ID NO: ComponentSEQ ID NO: ALG.APV-178 (scFv-Fc-scFv Format) HCDR1 2 HCDR1 30 HCDR2 4HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10 LCDR3 12LCDR3 36 V_(H) 14 V_(H) 38 V_(L) 16 V_(L) 40 scFv 110 scFv 118ALG.APV-179, ALG.APV-209, ALG.APV-222 (scFv-Fc-scFv Format) HCDR1 2HCDR1 30 HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 42 LCDR2 10LCDR2 10 LCDR3 12 LCDR3 36 V_(H) 14 V_(H) 38 V_(L) 16 V_(L) 44 scFv 110scFv 120 ALG.APV-187, ALG.APV-210, ALG.APV-223 (scFv-Fc-scFv Format)HCDR1 2 HCDR1 30 HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 42LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V_(H) 14 V_(H) 46 V_(L) 16 V_(L) 48scFv 110 scFv 122 ALG.APV-191 (scFv-Fc-scFv Format) HCDR1 18 HCDR1 30HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 42 LCDR2 10 LCDR2 10LCDR3 12 LCDR3 36 V_(H) 20 V_(H) 46 V_(L) 22 V_(L) 50 scFv 112 scFv 124ALG.APV-196 (scFv-Fc-scFv Format) HCDR1 24 HCDR1 52 HCDR2 4 HCDR2 32HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 54 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36V_(H) 26 V_(H) 56 V_(L) 16 V_(L) 58 scFv 114 scFv 126 ALG.APV-198(scFv-Fc-scFv Format) HCDR1 2 HCDR1 52 HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34LCDR1 8 LCDR1 54 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V_(H) 14 V_(H) 56V_(L) 16 V_(L) 58 scFv 110 scFv 126 ALG.APV-199 (scFv-Fc-scFv Format)HCDR1 2 HCDR1 60 HCDR2 4 HCDR2 62 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 54LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V_(H) 14 V_(H) 64 V_(L) 16 V_(L) 58scFv 110 scFv 128 ALG.APV-006 (scFv-Fc-scFv Format) HCDR1 24 HCDR1 30HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10LCDR3 12 LCDR3 36 V_(H) 28 V_(H) 38 V_(L) 16 V_(L) 68 scFv 116 scFv 130ALG.APV-010 (scFv-Fc-scFv Format) HCDR1 24 HCDR1 30 HCDR2 4 HCDR2 32HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36V_(H) 28 V_(H) 38 V_(L) 16 V_(L) 68 scFv 116 scFv 170 ALG.APV-014(scFv-Fc-scFv Format) HCDR1 24 HCDR1 30 HCDR2 4 HCDR2 32 HCDR3 6 HCDR334 LCDR1 8 LCDR1 8 LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V_(H) 28 V_(H) 46V_(L) 16 V_(L) 70 scFv 116 scFv 132 ALG.APV-018 (scFv-Fc-scFv Format)HCDR1 24 HCDR1 30 HCDR2 4 HCDR2 32 HCDR3 6 HCDR3 34 LCDR1 8 LCDR1 8LCDR2 10 LCDR2 10 LCDR3 12 LCDR3 36 V_(H) 28 V_(H) 46 V_(L) 16 V_(L) 70scFv 116 scFv 134

In some embodiments, the anti-5T4×anti-4-1BB molecules described hereinmay comprise an amino acid sequence that is at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, at least about 99.5%, or 100% identicalto an amino acid sequence selected from the group consisting of SEQ IDNOs: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174,and 176. In some embodiments, the anti-5T4×anti-4-1BB moleculesdescribed herein may comprise an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 172, 174, and 176. Exemplary amino acid and nucleic acidsequences for the anti-5T4×anti-4-1BB molecules described herein areprovided in Tables 10 and 11. In each of Tables 10 and 11,immunoglobulin Fc domains (hinge-CH1-CH2) are indicated in underlinedtext, and binding domain linker sequences are indicated in bolded text.

TABLE 10 Exemplary anti-5T4 x anti-4-1BB Molecule DNA Sequences DNAConstruct DNA SEQ SEQ ID ALG.APV-178ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 135CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGGGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTATTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCCAGGGCACAAAGCTGGAGATCAAGCGC ALG.APV-179ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 137CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGGGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCCAGGGCACAAAGCTGGAGATCAAGCGC ALG.APV-187ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 139CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGTGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAGCGC ALG.APV-191ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 141CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTGATTACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCACGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCACAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCTCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGTGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAGAGC ALG.APV-196ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 143CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCTCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCGACTTCGAGAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGTGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGGAGCGCCCTGAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAGCGC ALG.APV-198ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 145CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCGACTTCGAGAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGTGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGGAGCGCCCTGAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAGCGC ALG.APV-199ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 147CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCGACTTCGACAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGTGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGGGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGGAGCGCCCTGAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAGCGC ALG.APV-208ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 175CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTTCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGGGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTATTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCCAGGGCACAAAGCTGGAGATCAAGCGC ALG.APV-209ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 171 ALG.APV-222CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTTCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGGGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCCAGGGCACAAAGCTGGAGATCAAGCGC ALG.APV-210ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 173 ALG.APV-223CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTCACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCATGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGGGGTGGAGGTTCCGGAGGAGGCGGATCAGGTGGAGGCGGAAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTACAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTTCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAAGTGCAGCTGCTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCCTGAGGCTGAGCTGCGCTGCCTCCGGCTTCACATTCAGCAGCTATGCTATGAGCTGGGTGAGGCAAGCCCCTGGAAAGTGCCTGGAGTGGGTGTCCGCTATCTCCGGCAGCGGCGGAAGCACCTACTACGCTGACTCCGTCAAGGGCAGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAATAGCCTCAGGGCTGAAGACACCGCTGTGTACTACTGCGCCAGGTACTATGGCGGCTACTACTCCGCCTGGATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGGCGGAGGCGGCTCCGGAGGCGGTGGCTCCGGAGGAGGCGGAAGCGGAGGAGGAGGCTCCGATATTCAGATGACACAGTCCCCTAGCTCCCTGTCCGCCAGCGTGGGAGATCGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCTCCAGCTTCTTAAACTGGTACCAGCAGAAGCCTGGAAAGGCTCCCAAGCTGCTGATCTACGCCGCTTCCAGCCTCCAGAGCGGCGTGCCTAGCAGGTTCTCCGGCTCCGGAAGCGGAACAGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTCCGCTACCTACTACTGCCAGCAGACCTATGGCTACCTGCACACCTTCGGCTGCGGCACAAAGCTGGAGATCAAGCGC ALG.APV-006ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 149CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGAGGCTCCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCTCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACGGTGGTTACTACTCTGCTTGGATGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGCGGTGGAGGCAGCGGTGGGGGTGGGTCTGGAGGCGGTGGCAGTGGCGGCGGAGGCTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGACTTACGGTTACCTGCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCA ALG.APV-010ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 151CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGAGGCTCCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCTCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGACTTACGGTTACCTGCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGGCGGTGGAGGCAGCGGTGGGGGTGGGTCTGGAGGCGGTGGCAGTGGCGGCGGAGGCTCTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACGGTGGTTACTACTCTGCTTGGATGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA ALG.APV-014ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 153CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGAGGCTCCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCTCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGTGCCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACGGTGGTTACTACTCTGCTTGGATGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGCGGTGGAGGCAGCGGTGGGGGTGGGTCTGGAGGCGGTGGCAGTGGCGGCGGAGGCTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGACTTACGGTTACCTGCACACTTTTGGCTGCGGGACCAAGCTGGAGATCAAATCA ALG.APV-018ATGGAAGCACCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCA 155CCGGTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGAGGCTCCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCTCGAGTGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAATACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT TCCGGAGGTGGCGGTTCGGGAGGTGGCGGGTCAGGAGGTGGGGGATCCCCTTCAGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGACTTACGGTTACCTGCACACTTTTGGCTGCGGGACCAAGCTGGAGATCAAAGGCGGTGGAGGCAGCGGTGGGGGTGGGTCTGGAGGCGGTGGCAGTGGCGGCGGAGGCTCTGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGTGCCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACGGTGGTTACTACTCTGCTTGGATGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

TABLE 11 Exemplary anti-5T4 x anti-4-1BB Molecule Amino Acid SequencesAA Construct AA Sequence SEQ ID ALG.APV-178MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMY 136WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIKR ALG.APV-179MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMY 138WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIKR ALG.APV-187MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMY 140WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGCGTKLEIKR ALG.APV-191MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMY 142WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKS ALG.APV-196MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMY 144WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKR ALG.APV-198MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMY 146WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKR ALG.APV-199MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMY 148WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKCLEWVSAISGRGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKR ALG.APV-208MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMY 176WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIKR ALG.APV-209MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMY 172 ALG.APV-222WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGQGTKLEIKR ALG.APV-210MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSHGSMY 174 ALG.APV-223WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISHDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQTYGYLHTFGCGTKLEIKR ALG.APV-006MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMY 150WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIKS ALG.APV-010MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMY 152WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSGGGGSGGGGSGGGGSPSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS ALG.APV-014MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMY 154WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKS ALG.APV-018MEAPAQLLFLLLLWLPDTTGEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMY 156WVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIKSSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSGGGGSGGGGSGGGGSPSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS

Polynucleotides and Methods of Protein Expression

The disclosure also includes nucleic acids (e.g., DNA or RNA) encodingthe polypeptides of the present disclosure (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) or one or more polypeptide chains of a polypeptide asdescribed herein. Nucleic acids of the disclosure include nucleic acidshaving a region that is substantially identical to a polynucleotide aslisted in Tables 1-10, infra. In certain embodiments, a nucleic acid inaccordance with the present disclosure has at least 80%, typically atleast about 90%, and more typically at least about 95% or at least about98% identity to a polypeptide-encoding polynucleotide as listed inTables 1-10, wherein the nucleic acid encodes a polypeptide that is amultispecific polypeptide in the format scFv-Fc-scFv and wherein theencoded polypeptide comprises one or more of (1) a Y to F substitutionin the LCDR1 at position 99 of the anti-5T4 V_(L); (2) a F to Ssubstitution in the FR3 at position 148 of the anti-5T4 V_(L); (3) a Yto H substitution in the HCDR1 at position 150 of the anti-4-1BB V_(H);and (4) an R to H substitution in the FR3 at position 127 of theanti-4-1BB V_(H). Nucleic acids of the disclosure also includecomplementary nucleic acids. In some instances, the sequences will befully complementary (no mismatches) when aligned. In other instances,there can be up to about a 20% mismatch in the sequences. In someembodiments of the disclosure are provided nucleic acids encoding bothfirst and second polypeptide chains of a bispecific protein of thedisclosure. The nucleic acid sequences provided herein can be exploitedusing codon optimization, degenerate sequence, silent mutations, andother DNA techniques to optimize expression in a particular host, andthe present disclosure encompasses such sequence modifications.

The disclosure relates to an isolated nucleic acid molecule encodingpolypeptides of the present disclosure (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof), wherein said nucleic acid molecule comprises a nucleotidesequence selected from SEQ ID NOs: 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 171, 173, and 175.

Polynucleotide molecules comprising a desired polynucleotide sequenceare propagated by placing the molecule in a vector. Viral and non-viralvectors are used, including plasmids. The choice of plasmid will dependon the type of cell in which propagation is desired and the purpose ofpropagation. Certain vectors are useful for amplifying and making largeamounts of the desired DNA sequence. Other vectors are suitable forexpression in cells in culture. Still other vectors are suitable fortransfer and expression in cells in a whole animal or person. The choiceof appropriate vector is well within the skill of the art. Many suchvectors are available commercially. The partial or full-lengthpolynucleotide is inserted into a vector typically by means of DNAligase attachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

For expression, an expression cassette or system may be employed. Toexpress a nucleic acid encoding a polypeptide disclosed herein, anucleic acid molecule encoding the polypeptide, operably linked toregulatory sequences that control transcriptional expression in anexpression vector, is introduced into a host cell. In addition totranscriptional regulatory sequences, such as promoters and enhancers,expression vectors can include translational regulatory sequences and amarker gene which is suitable for selection of cells that carry theexpression vector. The gene product encoded by a polynucleotide of thedisclosure is expressed in any convenient expression system, including,for example, bacterial, yeast, insect, amphibian and mammalian systems.In the expression vector, the polypeptide-encoding polynucleotide islinked to a regulatory sequence as appropriate to obtain the desiredexpression properties. These can include promoters, enhancers,terminators, operators, repressors, and inducers. The promoters can beregulated (e.g., the promoter from the steroid inducible pIND vector(Invitrogen)) or constitutive (e.g., promoters from CMV, SV40,Elongation Factor, or LTR sequences). These are linked to the desirednucleotide sequence using the techniques described above for linkage tovectors. Any techniques known in the art can be used. Accordingly, theexpression vector will generally provide a transcriptional andtranslational initiation region, which can be inducible or constitutive,where the coding region is operably linked under the transcriptionalcontrol of the transcriptional initiation region, and a transcriptionaland translational termination region.

An expression cassette (“expression unit”) can be introduced into avariety of vectors, e.g., plasmid, BAC, YAC, bacteriophage such aslambda, P1, M13, etc., plant or animal viral vectors (e.g.,retroviral-based vectors, adenovirus vectors), and the like, where thevectors are normally characterized by the ability to provide selectionof cells comprising the expression vectors. The vectors can provide forextrachromosomal maintenance, particularly as plasmids or viruses, orfor integration into the host chromosome. Where extrachromosomalmaintenance is desired, an origin sequence is provided for thereplication of the plasmid, which can be low- or high copy-number. Awide variety of markers are available for selection, particularly thosewhich protect against toxins, more particularly against antibiotics. Theparticular marker that is chosen is selected in accordance with thenature of the host, where, in some cases, complementation can beemployed with auxotrophic hosts. Introduction of the DNA construct canuse any convenient method, including, e.g., conjugation, bacterialtransformation, calcium-precipitated DNA, electroporation, fusion,transfection, infection with viral vectors, biolistics, and the like.The disclosure relates to an expression vector comprising a nucleic acidsegment, wherein said nucleic acid segment may comprise a nucleotidesequence selected from SEQ ID NOs: 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 171, 173, and 175.

Accordingly, proteins for use within the present disclosure can beproduced in genetically engineered host cells according to conventionaltechniques. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells(including cultured cells of multicellular organisms), particularlycultured mammalian cells. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook and Russell, Molecular Cloning: A LaboratoryManual (3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001), and Ausubel et al., Short Protocols in MolecularBiology (4th ed., John Wiley & Sons, 1999).

For example, for recombinant expression of a homodimeric binding proteincomprising two identical binding polypeptides as described herein, anexpression vector will generally include a nucleic acid segment encodingthe binding polypeptide, operably linked to a promoter. For recombinantexpression of a heterodimeric binding protein comprising different firstand second polypeptide chains, the first and second polypeptide chainscan be co-expressed from separate vectors in the host cell forexpression of the entire heterodimeric protein. Alternatively, for theexpression of heterodimeric binding proteins the first and secondpolypeptide chains are co-expressed from separate expression units inthe same vector in the host cell for expression of the entireheterodimeric protein. The expression vector(s) are transferred to ahost cell by conventional techniques, and the transfected cells are thencultured by conventional techniques to produce the encodedpolypeptide(s) to produce the corresponding binding proteins (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/or multispecific binding proteins thereof).

To direct a recombinant protein into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence) isprovided in the expression vector. The secretory signal sequence can bethat of the native form of the recombinant protein, or can be derivedfrom another secreted protein or synthesized de novo. The secretorysignal sequence is operably linked to the polypeptide-encoding DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences can be positioned elsewherein the DNA sequence of interest (see, e.g., U.S. Pat. Nos. 5,037,743 and5,143,830).

Cultured mammalian cells are suitable hosts for production ofrecombinant polypeptides and proteins of the present disclosure (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) for use within the presentdisclosure. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., supra), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed by, for example, U.S. Pat. Nos. 4,713,339; 4,784,950;4,579,821; and 4,656,134. Examples of suitable mammalian host cellsinclude African green monkey kidney cells (Vero; ATCC CRL 1587), humanembryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidneycells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidneycells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCCCCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, Utah); see also, e.g.,Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitarycells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells(H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1;ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. Strong transcription promoters can be used, such aspromoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No.4,956,288. Other suitable promoters include those from metallothioneingenes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus majorlate promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants.” Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.”Exemplary selectable markers include a gene encoding resistance to theantibiotic neomycin, which allows selection to be carried out in thepresence of a neomycin-type drug, such as G-418 or the like; the gptgene for xanthine-guanine phosphoribosyl transferase, which permits hostcell growth in the presence of mycophenolic acid/xanthine; and markersthat provide resistance to zeocin, bleomycin, blastocidin, andhygromycin (see, e.g., Gatignol et al., Mol. Gen. Genet. 207:342, 1987;Drocourt et al., Nucl. Acids Res. 18:4009, 1990). Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.An exemplary amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J Biosci. (Bangalore) 11:47-58, 1987.Transformation of insect cells and production of foreign polypeptidestherein is disclosed in U.S. Pat. No. 5,162,222 and PCT Publication No.WO 94/06463.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See King and Possee, The Baculovirus Expression System: A LaboratoryGuide (Chapman & Hall, London); O'Reilly et al., Baculovirus ExpressionVectors: A Laboratory Manual (Oxford University Press., New York 1994);and Baculovirus Expression Protocols. Methods in Molecular Biology(Richardson ed., Humana Press, Totowa, N.J., 1995). Recombinantbaculovirus can also be produced through the use of a transposon-basedsystem described by Luckow et al. (J. Virol. 67:4566-4579, 1993). Thissystem, which utilizes transfer vectors, is commercially available inkit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, Md.). Thetransfer vector (e.g., PFASTBAC1; Life Technologies) contains a Tn7transposon to move the DNA encoding the protein of interest into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990;Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk andRapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfervectors can include an in-frame fusion with DNA encoding a polypeptideextension or affinity tag as disclosed above. Using techniques known inthe art, a transfer vector containing a protein-encoding DNA sequence istransformed into E. coli host cells, and the cells are screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, such as Sf9 cells. Recombinant virus thatexpresses the protein or interest is subsequently produced. Recombinantviral stocks are made by methods commonly used in the art.

For protein production, the recombinant virus is used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HIGHFIVE™ cells; Invitrogen, Carlsbad, Calif.). See generally Glick andPasternak, Molecular Biotechnology, Principles & Applications ofRecombinant DNA (ASM Press, Washington, D.C., 1994). See also U.S. Pat.No. 5,300,435. Serum-free media are used to grow and maintain the cells.Suitable media formulations are known in the art and can be obtainedfrom commercial suppliers. The cells are grown up from an inoculationdensity of approximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells, atwhich time a recombinant viral stock is added at a multiplicity ofinfection (MOI) of 0.1 to 10, more typically near 3. Procedures used aregenerally described in available laboratory manuals (see, e.g., King andPossee, supra; O'Reilly et al., supra; Richardson, supra).

Fungal cells, including yeast cells, can also be used within the presentdisclosure to produce the polypeptides of the present disclosure (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof). Yeast species of in this regardinclude, e.g., Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, U.S. Pat. Nos. 4,599,311; 4,931,373; 4,870,008; 5,037,743;and 4,845,075. Transformed cells are selected by phenotype determined bythe selectable marker, commonly drug resistance or the ability to growin the absence of a particular nutrient (e.g., leucine). An exemplaryvector system for use in Saccharomyces cerevisiae is the POTl vectorsystem disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), whichallows transformed cells to be selected by growth in glucose-containingmedia. Suitable promoters and terminators for use in yeast include thosefrom glycolytic enzyme genes (see, e.g., U.S. Pat. Nos. 4,599,311;4,615,974; and 4,977,092) and alcohol dehydrogenase genes. See also U.S.Pat. Nos. 4,990,446; 5,063,154; 5,139,936; and 4,661,454. Transformationsystems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii, and Candida maltosa are known in the art. See, e.g.,Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986; U.S. Pat. No.4,882,279; and Raymond et al., Yeast 14:11-23, 1998. Aspergillus cellscan be utilized according to the methods of McKnight et al., U.S. Pat.No. 4,935,349. Methods for transforming Acremonium chrysogenum aredisclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods fortransforming Neurospora are disclosed by Lambowitz, U.S. Pat. No.4,486,533. Production of recombinant proteins in Pichia methanolica isdisclosed in U.S. Pat. Nos. 5,716,808; 5,736,383; 5,854,039; and5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus, and other genera are also useful host cells within thepresent disclosure to produce, for example, 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof including anti-5T4×anti-4-1BB molecules. Techniques fortransforming these hosts and expressing foreign DNA sequences clonedtherein are well-known in the art (see, e.g., Sambrook and Russell,supra). When expressing a recombinant protein in bacteria such as E.coli, the protein can be retained in the cytoplasm, typically asinsoluble granules, or can be directed to the periplasmic space by abacterial secretion sequence. In the former case, the cells are lysed,and the granules are recovered and denatured using, for example,guanidine isothiocyanate or urea. The denatured protein can then berefolded and dimerized by diluting the denaturant, such as by dialysisagainst a solution of urea and a combination of reduced and oxidizedglutathione, followed by dialysis against a buffered saline solution. Inthe alternative, the protein can be recovered from the cytoplasm insoluble form and isolated without the use of denaturants. The protein isrecovered from the cell as an aqueous extract in, for example, phosphatebuffered saline. To capture the protein of interest, the extract isapplied directly to a chromatographic medium, such as an immobilizedantibody or heparin-Sepharose column. Secreted proteins can be recoveredfrom the periplasmic space in a soluble and functional form bydisrupting the cells (by, for example, sonication or osmotic shock) torelease the contents of the periplasmic space and recovering theprotein, thereby obviating the need for denaturation and refolding.Antibodies, including single-chain antibodies, can be produced inbacterial host cells according to known methods. See, e.g., Bird et al.,Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883, 1988; and Pantoliano et al., Biochem. 30:10117-10125,1991.

Transformed or transfected host cells to produce the polypeptides andproteins of the present disclosure (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof) are cultured according to conventional procedures in a culturemedium containing nutrients and other components required for the growthof the chosen host cells. A variety of suitable media, including definedmedia and complex media, are known in the art and generally include acarbon source, a nitrogen source, essential amino acids, vitamins andminerals. Media can also contain such components as growth factors orserum, as required. The growth medium will generally select for cellscontaining the exogenously added DNA by, for example, drug selection ordeficiency in an essential nutrient which is complemented by theselectable marker carried on the expression vector or co-transfectedinto the host cell.

The proteins and polypeptides of the present disclosure (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) may be purified by conventionalprotein purification methods, typically by a combination ofchromatographic techniques. See generally Affinity Chromatography:Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden,1988); Scopes, Protein Purification: Principles and Practice(Springer-Verlag, New York 1994). Proteins comprising an immunoglobulinFc region can be purified by affinity chromatography on immobilizedprotein A or protein G. Additional purification steps, such as gelfiltration, can be used to obtain the desired level of purity or toprovide for desalting, buffer exchange, and the like.

Compositions and Methods of Use

The present disclosure provides methods for treating a subject with adisorder characterized by expression of 5T4. Generally, such methodsinclude administering to a subject in need of such treatment a proteinthe present disclosure (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof). In someembodiments, a 5T4-binding polypeptide, 4-1BB-binding polypeptide,and/or multispecific binding proteins thereof (e.g., ananti-5T4×anti-4-1BB molecule) does not induce or induces minimalantibody-dependent cell-mediated cytotoxicity (ADCC) activity and/orcomplement-dependent cytotoxicity (CDC) activity.

In other embodiments, where the binding proteins (e.g., multispecificbinding proteins) comprise a second binding domain that specificallybinds an effector cell (e.g., to 4-1BB), the binding proteins and/ormultispecific binding proteins result in enhanced effector cellactivation against 5T4-expressing cells in the subject. For example, insome embodiments, the binding proteins and/or multispecific bindingproteins result in increased effector cell proliferation, increasedeffector cell production of one or more cytokines (e.g., IFNγ, TNFα,IL-6, IL-8, IL-12, IL-lα, etc.), or increased expression of one or morecell-surface activation markers (e.g., CD137, MHC-II, or CD69).Activation of effector cells can be measured by a variety of means knownin the art including flow cytometry, immunofluorescence, andimmunohistochemistry to assess changes in cell-surface markerexpression, ELISA to assess production of cytokines and other factors,cell counts to assess proliferation, qPCR to assess changes in geneexpression, and the like.

In some embodiments, the multi-specific binding proteins describedherein exhibit enhanced effector cell activation compared to a secondmultispecific polypeptide of a different structure. For example, in someembodiments, the first multispecific polypeptide comprises a first scFvdomain that specifically binds to 5T4 and a second scFv thatspecifically binds to 4-1BB, together by a binding domain linker and animmunoglobulin Fc domain in the following configuration, fromamino-terminus to carboxyl-terminus: (i) the first scFv domain, (ii) thehinge region, (iii) the immunoglobulin constant region, (iv) the bindingdomain linker, and (v) the second scFv domain. In this example, a secondmultispecific binding protein comprises an IgG-scFv structure comprisingan anti-4-1BB antibody and an anti-5T4 scFv. In some embodiments, thefirst multispecific binding protein exhibits a statistically significantenhancement of effector cell activation compared to the secondmultispecific polypeptide. For example, in some embodiments, the firstmultispecific binding protein exhibits a lower EC₅₀ value in aJurkat/NF-κB reporter cell assay than observed for the secondmultispecific binding protein (See e.g., Example 10). In someembodiments, the EC₅₀ of the first multispecific binding protein isdecreased by about 2 fold, about 3 fold, about 4 fold, about 5 fold,about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold,or more compared to the EC₅₀ of the second multispecific bindingprotein. EC₅₀ values can be determined by non-linear regression analysisand other statistical methods known in the art.

In some embodiments, the first multispecific binding protein induces astatistically significant increase in effector cell proliferationcompared to the second multispecific polypeptide. For example, in someembodiments, the first multispecific binding protein induces astatistically significant increase in proliferation of primed, humanCD8+ T cells compared to the proliferation induced by the secondmultispecific binding protein (See e.g., Example 22). In someembodiments, the first multispecific binding protein induces an increasein effector cell proliferation of about 2 fold, about 3 fold, about 4fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9fold, about 10 fold, or more compared to the effector cell proliferationinduced by the the second multispecific binding protein.

In certain variations of the method of treating a subject with a proteinthe present disclosure (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof), thedisorder is a cancer. Exemplary cancers amenable to treatment inaccordance with a protein the present disclosure include, for example,breast cancer (e.g., triple negative breast cancer (TNBC)), pancreaticcancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer),hematologic malignancies (e.g., chronic lymphocytic leukemia (CLL),mantle cell leukemia (MCL) or acute lymphoblastic leukemia (ALL)), skincancer (e.g., squamous cell carcinoma or melanoma), adrenal cancer,bladder cancer, cervical cancer, renal cancer, gastric cancer, prostatecancer, thyroid cancer, liver cancer, uterine cancer, a tumor formed ona nerve cell or nerve cell sheath (e.g., neurofibroma), sarcoma,carcinoma or head and neck cancer. TNBC is defined as breast cancer withthe absence of staining for estrogen receptor, progesterone receptor,and HER2/neu. In some embodiments, a protein of the present disclosurecan be administered to a subject to treat mesothelioma in the subject.In one embodiment, a protein of the present disclosure can beadministered to a subject to treat a clear cell carcinoma in thesubject. In one embodiment, a protein of the present disclosure can beadministered to a subject to treat a striated muscle tumor in thesubject.

In a further embodiment, the disclosure encompasses a method forenhancing effector cell activation against a cell expressing 5T4, themethod comprising contacting said 5T4-expressing cell with a protein ofthe present disclosure (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof), whereinsaid contacting is under conditions whereby enhanced effector cellactivation against the 5T4-expressing cell is induced. In someembodiments, the disclosure relates to a method for enhancing effectorcell activation against a cell expressing 5T4, the method comprising:contacting said 5T4-expressing cell with a protein of the presentdisclosure (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides,and/or multispecific binding proteins thereof) comprising a firstbinding domain that specifically binds an epitope of human 5T4 and asecond binding domain that specifically binds 4-1BB (e.g., ananti-5T4×anti-4-1BB molecule); wherein said contacting is underconditions whereby enhanced effector cell activation against the5T4-expressing cell is induced. The disclosure encompasses a method forinducing effector cell dependent lysis of a cell expressing 5T4, themethod comprising: contacting said 5T4-expressing cell with a protein ofthe present disclosure (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof), whereinthe second binding domain specifically binds 4-1BB (e.g., ananti-5T4×anti-4-1BB molecule); and wherein said contacting is underconditions whereby enhanced effector cell activation of the5T4-expressing cell is induced, thereby resulting in the lysis of the5T4-expressing cell. In some embodiments, the disclosure relates to amethod for inducing effector cell dependent lysis of a cell expressing5T4, the method comprising: contacting said 5T4-expressing cell with aprotein of the present disclosure comprising a first binding domain thatspecifically binds an epitope of human 5T4 and a second binding domainthat specifically binds 4-1BB (e.g., an anti-5T4×anti-4-1BB molecule);wherein said contacting is under conditions whereby enhanced effectorcell activation of the 5T4-expressing cell is induced thereby resultingthe lysis of the 5T4-expressing cell.

The disclosure also encompasses proteins and polypeptides (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) for the manufacture of amedicament for treatment of a disorder (e.g., cancer) characterized byexpression of 5T4. In one embodiment, the protein or polypeptidecomprises an anti-5T4 and anti-4-1BB binding domain (e.g., ananti-5T4×anti-4-1BB molecule) and has enhanced effector cell activationactivity. In one embodiment, the disclosure provides proteins andpolypeptides (e.g., an anti-5T4×anti-4-1BB molecule) for use in treatinga disorder (e.g., cancer) characterized by expression of 5T4. In certainembodiments, the disclosure relates to a method for treating a disorderin a subject, wherein said disorder is characterized by expression of5T4, the method comprising administering to the subject atherapeutically effective amount of a protein or polypeptide of thepresent disclosure comprising a 5T4 binding domain that specificallybinds an epitope of human 5T4 (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof).

In some embodiments, the disclosure provides a method of treating apatient with a cancer, comprising administering to the patient apolypeptide comprising amino acid sequence set forth in herein (e.g., anamino acid sequence selected from the group consisting of SEQ ID NOs:136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, 174, and176). In further embodiments, the polypeptide comprises an Fc. Forexample, in some embodiments, the disclosure provides a method oftreating a patient with a cancer, comprising administering to thepatient a polypeptide comprising a 5T4-binding domain and an Fc, whereinthe polypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 172, 174, and 176. In still further embodiments, thedisclosure provides a method of treating a patient with a cancercomprising administering to the patient a polypeptide comprising a5T4-binding domain and a 4-1BB-binding domain, wherein the polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172,174, and 176. In one embodiment, the disclosure provides a method oftreating a patient with a cancer comprising administering to the patienta polypeptide comprising a 5T4-binding domain and a 4-1BB-bindingdomain, wherein the polypeptide comprises SEQ ID NO: 172. In oneembodiment, the disclosure provides a method of treating a patient witha cancer comprising administering to the patient a polypeptidecomprising a 5T4-binding domain and a 4-1BB-binding domain, wherein thepolypeptide comprises SEQ ID NO: 174.

In some embodiments, for treatment methods and uses described herein, aprotein or polypeptide described herein (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof) is delivered in a manner consistent with conventionalmethodologies associated with management of the disease or disorder forwhich treatment is sought. In accordance with the disclosure herein, atherapeutically effective amount of the protein or polypeptide isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent or treat the disease or disorder.

Subjects for administration of a protein of the present disclosure(e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) include patients at high riskfor developing a particular disorder characterized by 5T4 expression aswell as patients presenting with an existing such disorder. Typically,the subject has been diagnosed as having the disorder for whichtreatment is sought. Further, subjects can be monitored during thecourse of treatment for any change in the disorder (e.g., for anincrease or decrease in clinical symptoms of the disorder). Also, insome variations, the subject does not suffer from another disorderrequiring treatment that involves targeting 5T4-expressing cells.

In prophylactic applications, pharmaceutical compositions or medicantscomprising a protein of the present disclosure (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) are administered to a patient susceptible to, orotherwise at risk of, a particular disorder in an amount sufficient toeliminate or reduce the risk or delay the onset of the disorder. Intherapeutic applications, compositions or medicants comprising a proteinof the present disclosure are administered to a patient suspected of, oralready suffering from such a disorder in an amount sufficient to cure,or at least partially arrest, the symptoms of the disorder and itscomplications. An amount adequate to accomplish this is referred to as atherapeutically effective dose or amount. In both prophylactic andtherapeutic regimes, agents are usually administered in several dosagesuntil a sufficient response (e.g., inhibition of inappropriateangiogenesis activity) has been achieved. Typically, the response ismonitored and repeated dosages are given if the desired response startsto fade.

To identify subject patients for treatment according to the methods ofthe disclosure (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof), acceptedscreening methods can be employed to determine risk factors associatedwith specific disorders or to determine the status of an existingdisorder identified in a subject. Such methods can include, for example,determining whether an individual has relatives who have been diagnosedwith a particular disorder. Screening methods can also include, forexample, conventional work-ups to determine familial status for aparticular disorder known to have a heritable component. For example,various cancers are also known to have certain inheritable components.Inheritable components of cancers include, for example, mutations inmultiple genes that are transforming (e.g., Ras, Raf, EGFR, cMet, andothers), the presence or absence of certain HLA and killer inhibitoryreceptor (KIR) molecules, or mechanisms by which cancer cells are ableto modulate immune suppression of cells like NK cells and T-cells,either directly or indirectly (see, e.g., Ljunggren and Malmberg, NatureRev. Immunol. 7:329-339, 2007; Boyton and Altmann, Clin. Exp. Immunol.149:1-8, 2007). Toward this end, nucleotide probes can be routinelyemployed to identify individuals carrying genetic markers associatedwith a particular disorder of interest. In addition, a wide variety ofimmunological methods are known in the art that are useful to identifymarkers for specific disorder. For example, various ELISA immunoassaymethods are available and well-known in the art that employ monoclonalantibody probes to detect antigens associated with specific tumors.Screening can be implemented as indicated by known patient symptomology,age factors, related risk factors, etc. These methods allow theclinician to routinely select patients in need of the methods describedherein for treatment. In accordance with these methods, targetingpathological, 5T4-expressing cells can be implemented as an independenttreatment program or as a follow-up, adjunct, or coordinate treatmentregimen to other treatments.

For administration, a protein of the present disclosure (e.g.,5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) may be formulated as apharmaceutical composition. A pharmaceutical composition may comprise:(i) a 5T4-binding polypeptide, a 4-1BB-binding polypeptide, and/ormultispecific binding protein thereof (e.g., an anti-5T4×anti-4-1BBmolecule); and (ii) a pharmaceutically acceptable carrier, diluent orexcipient. A pharmaceutical composition comprising a 5T4-bindingpolypeptide, a 4-1BB-binding polypeptide, and/or multi specific bindingprotein thereof (e.g., an anti-5T4×anti-4-1BB molecule) can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic molecule is combined in a mixturewith a pharmaceutically acceptable carrier, diluent or excipient. Acarrier is said to be a “pharmaceutically acceptable carrier” if itsadministration can be tolerated by a recipient patient. Sterilephosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers, diluents or excipients arewell-known to those in the art. (See, e.g., Gennaro (ed.), Remington'sPharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).)Formulations can further include one or more excipients, preservatives,solubilizers, buffering agents, albumin to prevent protein loss on vialsurfaces, etc. In certain embodiments, a pharmaceutical compositioncomprises a 5T4-binding polypeptide, a 4-1BB-binding polypeptide, and/ormultispecific binding protein thereof (e.g., an anti-5T4×anti-4-1BBmolecule) that is a homodimer or a heterodimer. A “homodimer” may be adimer formed from two identical polypeptides (e.g., ananti-5T4×anti-4-1BB molecule as described herein).

A pharmaceutical composition comprising a polypeptide or proteindescribed herein may be formulated in a dosage form selected from thegroup consisting of: an oral unit dosage form, an intravenous unitdosage form, an intranasal unit dosage form, a suppository unit dosageform, an intradermal unit dosage form, an intramuscular unit dosageform, an intraperitoneal unit dosage form, a subcutaneous unit dosageform, an epidural unit dosage form, a sublingual unit dosage form, andan intracerebral unit dosage form. The oral unit dosage form may beselected from the group consisting of: tablets, pills, pellets,capsules, powders, lozenges, granules, solutions, suspensions,emulsions, syrups, elixirs, sustained-release formulations, aerosols,and sprays.

A pharmaceutical composition comprising polypeptide or protein describedherein (e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides,and/or multispecific binding proteins thereof) may be administered to asubject in a therapeutically effective amount. According to the methodsof the present disclosure, polypeptide or protein described herein(e.g., 5T4-binding polypeptides, 4-1BB-binding polypeptides, and/ormultispecific binding proteins thereof) can be administered to subjectsby a variety of administration modes, including, for example, byintramuscular, subcutaneous, intravenous, intra-atrial, intra-articular,parenteral, intranasal, intrapulmonary, transdermal, intrapleural,intrathecal, and oral routes of administration. For prevention andtreatment purposes, an agonist (e.g., 5T4-binding polypeptides,4-1BB-binding polypeptides, and/or multispecific binding proteinsthereof) can be administered to a subject in a single bolus delivery,via continuous delivery (e.g., continuous transdermal delivery) over anextended time period, or in a repeated administration protocol (e.g., onan hourly, daily, weekly, or monthly basis).

Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby determining effective dosages and administration protocols thatsignificantly reduce the occurrence or severity of the subject disorderin model subjects. Effective doses of the compositions of the presentdisclosure vary depending upon many different factors, including meansof administration, target site, physiological state of the patient,whether the patient is human or an animal, other medicationsadministered, whether treatment is prophylactic or therapeutic, as wellas the specific activity of the composition itself and its ability toelicit the desired response in the individual. Usually, the patient is ahuman, but in some diseases, the patient can be a nonhuman mammal.Typically, dosage regimens are adjusted to provide an optimumtherapeutic response, i.e., to optimize safety and efficacy.Accordingly, a therapeutically effective amount is also one in which anyundesired collateral effects are outweighed by the beneficial effects ofadministering a 5T4-binding polypeptide, a 4-1BB-binding polypeptide,and/or a multispecific binding protein thereof (e.g., ananti-5T4×anti-4-1BB molecule) as described herein. For administration ofa protein or polypeptide described herein, a dosage may range from about0.1 μg to 100 mg/kg or 1 μg/kg to about 50 mg/kg, and more usually 10 μgto 5 mg/kg of the subject's body weight. In more specific embodiments,an effective amount of the agent is between about 1 μg/kg and about 20mg/kg, between about 10 μg/kg and about 10 mg/kg, or between about 0.1mg/kg and about 5 mg/kg. Dosages within this range can be achieved bysingle or multiple administrations, including, e.g., multipleadministrations per day or daily, weekly, bi-weekly, or monthlyadministrations. For example, in certain variations, a regimen consistsof an initial administration followed by multiple, subsequentadministrations at weekly or bi-weekly intervals. Another regimenconsists of an initial administration followed by multiple, subsequentadministrations at monthly or bi-monthly intervals. Alternatively,administrations can be on an irregular basis as indicated by monitoringclinical symptoms of the disorder.

Dosage of the pharmaceutical composition comprising a polypeptide orprotein described herein (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof) can bevaried by the attending clinician to maintain a desired concentration ata target site. For example, if an intravenous mode of delivery isselected, local concentration of the agent in the bloodstream at thetarget tissue can be between about 0.01-50 nanomoles of the compositionper liter, sometimes between about 1.0 nanomole per liter and 10, 15, or25 nanomoles per liter depending on the subject's status and projectedmeasured response. Higher or lower concentrations can be selected basedon the mode of delivery, e.g., trans-epidermal delivery versus deliveryto a mucosal surface. Dosage should also be adjusted based on therelease rate of the administered formulation, e.g., nasal spray versuspowder, sustained release oral or injected particles, transdermalformulations, etc. To achieve the same serum concentration level, forexample, slow-release particles with a release rate of 5 nanomolar(under standard conditions) would be administered at about twice thedosage of particles with a release rate of 10 nanomolar.

The proteins and polypeptides described herein (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof) (e.g., an anti-5T4×anti-4-1BB molecule) may also beadministered at a daily dosage of from about 0.001 to about 10milligrams (mg) per kilogram (mpk) of body weight, preferably given as asingle daily dose or in divided doses about two to six times a day. Foradministration to a human adult patient, the therapeutically effectiveamount may be administered in doses in the range of 0.2 mg to 800 mg perdose, including but not limited to 0.2 mg per dose, 0.5 mg per dose, 1mg per dose, 5 mg per dose, 10 mg per dose, 25 mg per dose, 100 mg perdose, 200 mg per dose, and 400 mg per dose, and multiple, usuallyconsecutive daily doses may be administered in a course of treatment.The proteins and polypeptides described herein (e.g., 5T4-bindingpolypeptides, 4-1BB-binding polypeptides, and/or multispecific bindingproteins thereof such as an anti-5T4×anti-4-1BB molecule) can beadministered at different times of the day. In one embodiment theoptimal therapeutic dose can be administered in the evening. In anotherembodiment the optimal therapeutic dose can be administered in themorning. The total daily dosage of the proteins and polypeptidesdescribed herein thus can in one embodiment range from about 1 mg toabout 2 g, and often ranges from about 100 mg to about 1.5 g, and mostoften ranges from about 200 mg to about 1200 mg. In the case of atypical 70 kg adult human, the total daily dose of the anti-5T4therapeutic can range from about 2 mg to about 1200 mg and will oftenrange, as noted above, from about 0.2 mg to about 800 mg.

With particular regard to treatment of solid tumors, protocols forassessing endpoints and anti-tumor activity are well-known in the art.While each protocol may define tumor response assessments differently,the RECIST (Response evaluation Criteria in solid tumors) criteria iscurrently considered to be the recommended guidelines for assessment oftumor response by the National Cancer Institute (see Therasse et al., J.Natl. Cancer Inst. 92:205-216, 2000). According to the RECIST criteriatumor response means a reduction or elimination of all measurablelesions or metastases. Disease is generally considered measurable if itcomprises lesions that can be accurately measured in at least onedimension as ≥20 mm with conventional techniques or ≥10 mm with spiralCT scan with clearly defined margins by medical photograph or X-ray,computerized axial tomography (CT), magnetic resonance imaging (MRI), orclinical examination (if lesions are superficial). Non-measurabledisease means the disease comprises of lesions <20 mm with conventionaltechniques or <10 mm with spiral CT scan, and truly non-measurablelesions (too small to accurately measure). Non-measureable diseaseincludes pleural effusions, ascites, and disease documented by indirectevidence.

The criteria for objective status are required for protocols to assesssolid tumor response. Representative criteria include the following: (1)Complete Response (CR), defined as complete disappearance of allmeasurable disease; no new lesions; no disease related symptoms; noevidence of non-measurable disease; (2) Partial Response (PR) defined as30% decrease in the sum of the longest diameter of target lesions (3)Progressive Disease (PD), defined as 20% increase in the sum of thelongest diameter of target lesions or appearance of any new lesion; (4)Stable or No Response, defined as not qualifying for CR, PR, orProgressive Disease. (See Therasse et al., supra.)

Additional endpoints that are accepted within the oncology art includeoverall survival (OS), disease-free survival (DFS), objective responserate (ORR), time to progression (TTP), and progression-free survival(PFS) (see Guidance for Industry: Clinical Trial Endpoints for theApproval of Cancer Drugs and Biologics, April 2005, Center for DrugEvaluation and Research, FDA, Rockville, Md.)

Pharmaceutical compositions comprising the proteins and polypeptidesdescribed herein (e.g., 5T4-binding polypeptides, 4-1BB-bindingpolypeptides, and/or multispecific binding proteins thereof such as ananti-5T4×anti-4-1BB molecule) can be supplied as a kit comprising acontainer that comprises the pharmaceutical composition as describedherein. A pharmaceutical composition can be provided, for example, inthe form of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before inj ection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a pharmaceuticalcomposition. Such a kit can further comprise written information onindications and usage of the pharmaceutical composition.

The disclosure will be further clarified by the following examples,which are intended to be purely exemplary of the disclosure and in noway limiting.

EXAMPLES Example 1 Conversion of “1618” Anti-CD137 Monoclonal Antibodyto scFv Format and Generation of Bispecific Anti-CD137×Anti-5T4Molecules in ADAPTIR™ Format (scFv-Fc-scFv)

In order to generate a nucleotide sequence encoding a 1618 scFv,nucleotide sequences encoding the variable domains of the heavy chain(V_(H)) and light chain (V_(L)) (SEQ ID NO: 28 and SEQ ID NO: 16,respectively) of the 1618 anti-4-1BB monoclonal antibody weresynthesized and/or amplified from existing plasmid DNA and linkedtogether by a (Gly)₄Ser-based linker using standard molecular biologytechniques those described in, but not limited to e.g., PCT ApplicationPublication Nos. WO 2007/146968, WO 2010/040105 and WO 2010/003108; U.S.Patent Application Publication Nos. 2006/0051844; and U.S. Pat. No.7,166,707. The nucleotide sequence of the 1618 scFv was then fused to amodified human IgG1 Fc region sequence comprising point mutations toeliminate the effector function activities of the Fc region. Similarly,nucleotide sequences encoding the V_(H) and V_(L) domains of the 1210anti-5T4 monoclonal antibody (SEQ ID NOs: 38 and 68, respectively) werelinked together by an additional (Gly)₄Ser-based linker. The resultingbispecific molecules were expressed via transient transfection ofHEK-293 or Chinese Hamster Ovary (CHO) cells and purified fromconditioned media using Protein A affinity purification (ProA) and sizeexclusion chromatography (SEC). Protein purity was determined byanalytical SEC after each of the Protein A and SEC purification steps.Endotoxin levels were determined by using the Endosafe PTS instrumentaccording to the manufacturer's instructs to assure that the in vitroactivity assay results would not be confounded by the presence ofendotoxin. Each protein batch was buffer-exchanged into PBS as part ofthe SEC purification process, concentrated to 1 mg/mL, sterile-filtered,and stored at 4° C. until needed. Protein concentration was determinedfrom the absorbance at 280 nm and using the theoretical extinctioncoefficient.

In some instances, the bispecific molecules were comprised of 2 scFvsand 1 Fc domain (scFv-Fc-scFv) with the following structure, fromN-terminus to C-terminus: 1618 scFv-Fc-1210 scFv. Additional bispecificmolecules were generated in which the 1618 scFv was placed on theC-terminus of the construct and the 1210 scFv on the N-terminus (i.e.,1210 scFv-Fc-1618 scFv). However, molecules with this alternativeorientation had less desirable properties when evaluated compared to the1618 scFv-Fc-1210 scFv-Fc-scFv molecules.

Most, but not all, of the 1618-Fc-1210 scFv-Fc-scFv ADAPTIR™ molecules(e.g., ALG.APV-006, ALG.APV-010, ALG.APV-014, and ALG.APV-018) hadsignificantly improved expression levels when transiently transfectedinto HEK-293 cells compared to the Morrison format (i.e. ALG.APV-004).Table 12 contains representative data showing the improved expressionlevels of the ADAPTIR™ bispecific molecules (e.g., ALG.APV-006,ALG.APV-010, ALG.APV-014, and ALG.APV-018) versus the Morrisonbispecific constructs (ALG.APV-004). This was an unexpected result, asthe amino acid sequence of the 1618 and 1210 variable domains were notmodified to obtain this improvement. These results indicate that theADAPTIR™ bispecific molecules may be beneficial for manufacturing oftherapeutic protein drugs; higher expression levels are generallyconsidered beneficial for therapeutic protein drug manufacturing as thiscan provide lower cost of goods and other efficiencies in themanufacturing process, such as fewer production runs needed to meetmarket requirements.

In addition to the apparent improved expression in HEK-293, theaggregate levels quantified after Protein A purification of thebispecific molecule from conditioned media were significantly improvedin the ADAPTIR™ bispecifics (ALG.APV-006, ALG.APV-010, ALG.APV-014 andALG.APV-018) compared to the Morrison format bispecific (ALG.APV-004),as shown in Table 12.

TABLE 12 HEK-293 transient expression levels and post-Protein A purityof ADAPTIR ™ bispecifics vs. Morrison bispecifics Expression % PurityBispecific Level Analytical Construct Construct Distinction Format μg/mLSEC ALG.APV-004 1618 mAb-1210 V_(H)V_(L) Morrison 13.0 81.7w/stabilizing disulfide ALG.APV-006 1618 V_(H)V_(L) -Fc-1210 V_(H)V_(L)ADAPTIR ™ 25.5 94.4 ALG.APV-010 1618 V_(H)V_(L) -Fc-1210 V_(L)V_(H)ADAPTIR ™ 55.9 94.3 ALG.APV-014 1618 V_(H)V_(L) -Fc 1210 V_(H)V_(L)ADAPTIR ™ 18.9 89.2 w/stabilizing disulfide ALG.APV-018 1618 V_(H)V_(L)-Fc 1210 V_(L)V_(H) ADAPTIR ™ 25.8 89.1 w/stabilizing disulfide

The order of the V_(H) and V_(L) domains was also evaluated with boththe 1210 and 1618 scFvs (V_(H)-V_(L) or V_(L)-V_(H)) and it wasdetermined that the preferred orientation of the 1618 scFv wasV_(H)-V_(L), as the expression levels of scFv-Fc-scFv molecules weresignificantly reduced when the V_(L)-V_(H) orientation was utilized. Thepreferred orientation of the 1210 scFv was V_(H)-V_(L), which conferredimproved binding to 5T4-expressing cells and in vitro activity in aCD137 reporter assay when compared to bispecific molecules that werecomprised of the 1210 scFv in the V_(L)-V_(H) format as shown in Table13.

TABLE 13 Comparison of cell binding (EC₅₀) and in vitro activity in aCD137 reporter assay when the 1210 scFv is either in the V_(H)-V_(L) orV_(L)-V_(H) orientation Affinity to Human 5T4- expressing cells, EC₅₀(nM), CD137 Reporter Construct Construct Distinction measured by flowcytometry Assay EC₅₀ (nM) ALG.APV-006 1618 V_(H)V_(L) -Fc-1210V_(H)V_(L) 15.7 0.23 ALG.APV-010 1618 V_(H)V_(L) -Fc-1210 V_(L)V_(H)50.9 2.60

The length of the linker between the V_(H) and V_(L) domains of the 1618and 1210 scFvs was also evaluated. Changing the linker length connectingthe V_(H) and V_(L) to either a 4× or 3× repeat of the Gly4Ser linkerdid not appear to result in a significant difference in activity orstability (data not shown), so the 4× repeat was generally used unlessotherwise denoted.

Example 2 Optimization of the 1618 Anti-CD137 scFv and Anti-5T4 scFvBinding Domains

After initial characterization of ADAPTIR™ bispecific (ALG.APV-006), itwas desirable to increase 1) the binding affinities of the scFv bindingdomains to their respective targets; 2) the biophysical stability; and3) the in vitro activity. Improvements in these parameters are expectedto lead to improved cost of goods, ease of manufacturing and reducedclinical dosage amounts. For binding domain optimization, randommutagenesis phage libraries were generated for both the 1618 and 1210scFV using error-prone PCR. Briefly, each scFv was used as template inan error-prone PCR reaction using a commercial mutagenesis kit(GeneMorph II Random Mutagenesis Kit, Agilent Technologies) followingthe manufacturer's protocol. The epPCR products were digested, ligatedinto the phagemid-scFV vector, and transformed into E. coli SS320/M13K07competent cells to generate the phage libraries. Five rounds of panningwere performed on each library, using biotinylated 4-1BB or 5T4 ECD asantigen (for the 1618 or 1210 binding domains, respectively). Increasedstringency of panning was used for each successive round by decreasingthe antigen concentration and increasing the wash times. Following thefinal round of panning, phage output was plated and prepared for a bulkcloning of the scFv pool into the prepared ADAPTIR™ expression vector.Approximately 400 individual colonies were picked, phagemids isolatedand sequenced, and the purified DNA used in high-throughput small scale293 transient transfections (˜0.6 mL culture volume). 5-day clarifiedsupernatants were assayed for binding by Flow Cytometry and SPR. Thebest performing variants were then carried forward through a battery oftests, including cell binding, in vitro activity and various stabilityassays. These bispecific ADAPTIR™ variants were produced with changes toeither the 1210 or 1618 scFv, while keeping the other scFv as theunmodified parental sequence. This simplified the interpretation of thedata and allowed us to assess the impact of these single changes onbinding, activity and stability. Beneficial point mutations wereidentified, based on improvements to stability, affinity or bioactivity.These were then combined to produce an addition set of ADAPTIR DNAconstructs. These constructs incorporated multiple changes in either the1210 and/or 1618 scFv. Each construct was expressed by HEK-293 cells viatransient transfection in 250 mL culture volume and purified using ProAand SEC steps. Final protein purity was verified by analytical SEC andthe endotoxin levels was determined by using the Endosafe PTS instrumentaccording to the manufacturer's instructs to assure that the in vitroactivity assay results would not be confounded by the presence ofendotoxin. The resulting protein was buffer-exchanged into PBS as partof the SEC purification process, concentrated to 1 mg/mL,sterile-filtered, and stored at 4° C. until needed. Proteinconcentration was determined from the absorbance at 280 nm and using thetheoretical extinction coefficient.

Example 3 Evaluation of Phage-Derived Optimized Variants

Several beneficial amino acid changes to 1210 and 1618 scFvs wereidentified as part of the phage panning efforts, when compared to theparent ALG.APV-006 construct. One measure of protein colloidal stabilityis to examine the amount of protein that is precipitated out of solutionwhen ammonium sulfate is added to a final concentration of ˜1M. Underthese conditions, the parent construct ALG.APV-006 lost nearly 89% ofthe protein in solution (Table 14). In comparison, the changesrepresented by ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150 allshow reduced protein loss under these conditions, suggesting that theindividual amino acid changes made in these constructs improved thecolloidal stability of the ADAPTIR™ bispecifics. Another measure ofprotein stability is to look at the impact of applying a shear force toa protein sample and examining that sample for loss due to precipitationor formation of soluble, aggregated forms of the bispecific. Whenassessed in a shear assay in which the protein solution was placed in a96-well plate and shaken at 2,000 RPM for two hours, the ALG.APV-127mutation significantly reduced the amount of protein loss, while theother variants had minimal effect or slightly exacerbated loss (Table14). Another measure of thermostability can be determined by usingdifferential scanning calorimetry (DSC) or differential scanningfluorimetry (DSF) to determine the mid-point temperature of the meltingcurve, otherwise known at Tm. Upon determination of the Tm of the 1618and 1210 variants, we noted that the ALG.APV-127 and ALG.APV-150variants increased the Tm of the 1618 scFv by three to four degrees,when compared to the ALG.APV-006 parental sequence (Table 14). Anincrease in the value of Tm can generally be interpreted as animprovement in a folded protein's stability, as it means that theprotein is more resistant to heat-induced unfolding/denaturation.ALG.APV-148 increased the thermostability of the 1210 scFv, as the Tmwas increased nearly three degrees compared to the parent 1210 sequenceused in ALG.APV-006.

Various combinations of the ALG.APV-099, ALG.APV-127, ALG.APV-148, andALG.APV-150 mutations were evaluated to see if combining mutationsprovided additive benefit to the biophysical stability of the ADAPTIR™bispecfic constructs. These were expressed and purified using the samemethod and evaluated in the same assays as the individual pointmutations. The results in Table 14 show that the combination ofmutations included in ALG.APV-178 and ALG.APV-179, as representativeexamples, resulted in stability increases that were generally equivalentor better than that obtained from individual changes.

TABLE 14 Biophysical assessment data for non-optimized and optimizedADAPTIR ™ bispecific constructs 1M Ammonium Sulfate Shear Stress, Tm (°C.) Tm (° C.) Construct Modified Domain Solubility, % Protein Loss %Protein Loss 1618 scFv 1210 scFv ALG.APV-006 Parent Sequence 89 64 56.170.5 of 1210 and 1618 ALG.APV-099 1210 67 80 57 70.5 ALG.APV-127 1618 5532 60.4 71.0 ALG.APV-148 1210 24 76 56.9 73.0 ALG.APV-150 1618 56 7158.9 70.8 ALG.APV-178 1210 and 1618 18.7 24.5 61.7 72.3 ALG.APV-179 1210and 1618 28.9 26.5 61.7 72.3

In addition to the stability assessments that were performed, thebinding affinity of ALG.APV-006 and the phage derived variants weredetermined by Surface Plasmon Resonance (SPR) using a Biacore T-200,using the recombinant extracellular domains of human 5T4 and humanCD137. ALG.APV-127 and ALG.APV-150 modifications led to a significantlytighter binding affinity to CD137 (Table 15). Similarly, it was observedthat ALG.APV-099 binds significantly more tightly than ALG.APV-006 (13vs. 149 nM, respectively).

TABLE 15 Summary of binding and activity assessments for non-optimizedand optimized ADAPTIR ™ bispecific constructs Affinity to Human Affinityto Human CD137by SPR 5T4 by SPR In vitro activity Construct ModifiedDomain KD (nM) KD (nM) EC₅₀ (nM) ALG.APV-006 Parent Sequences 128  1490.23 ALG.APV-099 1210 N/A  13 0.09 ALG.APV-127 1618 58 N/A 0.03ALG.APV-148 1210 N/A 198 0.05 ALG.APV-150 1618 97 N/A 0.03

The binding of the ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150variants to CD137 and 5T4 expressed on the surface of cells was comparedto the binding of ALG.APV-006. In spite of the improved affinity forsoluble CD137 or 5T4 displayed by some of these mutations, noappreciable difference was observed in on-cell binding experiments (datanot shown). To compare the effectiveness of the phage-display derivedvariants at inducing target-dependent activation of CD137, the variantswere compared in a CD137 reporter assay.

CD137 reporter assay: Jurkat/CD137 transfectants carrying a luciferasereporter gene under the control of an NF-κB promoter (Promega) werecultured according to the manufacturer's protocols. Jurkat/NF-κBreporter cells were cultured with CHO-K1 cells transfected with human5T4, at approximately 15,000 reporter cells to 30,000 target cells in96-well plates. Concentrations of bispecific molecules with finalconcentration ranging from 10 nM to 0.002 nM were added. Cells werecultured in a total volume of 100 μL of RPMI 1640 media supplementedwith 1% fetal bovine serum, sodium pyruvate, antibiotics andnon-essential amino acids. Plates were incubated at 37° C., 5% CO₂ inhumidified incubators for 5 to 6 hours. One hundred microliters ofBio-Glow buffer (Promega) was added to each well and incubated for 5 to10 minutes before measuring fluorescence. Luminescence was measured in aMicroBeta² 2450 Microplate Counter (Perkin Elmer). Nonlinear regressionanalysis to determine EC₅₀ values was performed in GraphPad Prism 6®graphing and statistics software.

FIG. 1 shows that all the constructs displayed agonistic function in thepresence of 5T4(+) cells; no reporter activity was observed in theabsence of 5T4(+) cells (not shown). The constructs ALG.APV-099,ALG.APV-127, ALG.APV-148 and ALG.APV-150 displayed better agonistfunction (lower EC₅₀ values) than the original ALG.APV-006 construct.Every point in the curve represents the average of duplicate wells. TheMorrison format molecule ALG.APV-004 was included for comparison. The yaxis shows values in relative fluorescence units (RLU) represented aspercent of maximum fluorescence.

Due to the improvements obtained with the single point mutationsrepresented by ALG.APV-099, ALG.APV-127, ALG.APV-148 and ALG.APV-150,these were combined in various ways in order to determine if thecharacteristics of the individuals would have additive benefit whenincluded in the same protein. These unique mutations were evaluated incombination to derive variants of 1618 and 1210 scFvs with significantlyimproved properties such as ALG.APV-178, ALG.APV-179, ALG.APV-187,ALG.APV-191, ALG.APV-196, ALG.APV-198 and ALG.APV-199. To generate theALG.APV-191, ALG.APV-196, ALG.APV-198 and ALG.APV-199 constructs, thesingle point mutations were further combined with the LO1, LO2, and LO11mutations described in Examples 22 and 23 of PCT Application No.PCT/EP2017/059656. Further, targeted phage libraries were created afteranalyzing regions of the 1618 and 1210 scFvs to identify regions thatmay contribute to instability. However, panning these libraries did notyield scFvs with improved properties (data not shown).

Example 4 Evaluation of Stabilizing Disulfide Bonds

Point mutations to incorporate an additional cysteine residue into theV_(H) and V_(L) domains of both the 1618 and 1210 scFvs were made. Whenthese bispecific molecules are expressed, these cysteines react to forma disulfide bond and can act to increase the stability of the scFv andconfer beneficial properties to improve the manufacturing and storage ofthese products. Experiments were performed showing that when optimizedbispecific molecules with the stabilizing disulfide in the 1210 scFv arestored for one week at 4 or 25° C., or submitted to three freeze-thawcycles between −80° C. and room temperature, they maintain their puritybetter than bispecific molecules without the disulfide. Representativedata is shown in Table 16. Addition of a stabilizing disulfide bond tothe 1618 scFv was also evaluated. While the disulfide appeared to confera stability benefit, the Biacore binding data suggested that there washeterogeneity in the sample leading to atypical binding kinetics (datanot shown).

TABLE 16 Improvement in storage and freeze-thaw stability in bispecificmolecules containing a stabilizing disulfide bond in the 1210 scFvChange in % Purity Is stabilizing Decrease in % Decrease in % after 3Freeze-Thaw disulfide in Purity Purity Cycles from −80° C. Construct1210 scFv? Day 7 @ 4° C. Day 7 @ 25° C. to Room Temperature ALG.APV-178No 2.4 1.9 4 ALG.APV-179 No 1.5 1.7 4 ALG.APV-187 Yes 0.1 0.1 1ALG.APV-191 Yes 0.0 0.0 2 ALG.APV-196 Yes 0.0 0.1 2 ALG.APV-198 Yes 0.10.1 2 ALG.APV-199 Yes 0.0 0.1 1

The improvement in bispecific molecule stability with the inclusion of astabilizing disulfide bond in the 1210 scFv was also evident in asubsequent evaluation performed at 40° C. Two constructs, ALG.APV-209and ALG.APV-210 differ by the addition of a stabilizing disulfide in the1210 scFv of ALG.APV-210. After one week at this elevated temperature,ALG.APV-210 formed significantly less aggregated material (Table 17).

TABLE 17 Improved 40° C. stability data for a bispecific molecule with astabilizing disulfide bond in the 1210 scFv Is stabilizing disulfideDecrease in % Purity Construct in 1210 scFv? Day 7 @ 40° C. ALG.APV-209No 2.6 ALG.APV-210 Yes 0.2

While the inclusion of the disulfide bond in 1210 provided significantbenefit in storage stability, the other stability parameters that wereassessed did not detect a significant difference between the twoconstructs (Table 18). Both constructs performed significantly better inthese assays as compared to the parent bispecific molecule ALG-006(Table 14).

TABLE 18 Comparision of ALG.APV-209 to ALG.APV-210 stability data Isstabilizing 1M Ammonium Shear Stress, disulfide in Modified SulfateSolubility, % Protein Tm (° C.) Tm (° C.) Construct 1210 scFv? Domain %Protein Loss Loss 1618 scFv 1210 scFv ALG.APV-209 No 1210 and 1618 16.222.1 61.2 72.7 ALG.APV-210 Yes 1210 and 1618 13.1 23.1 61.5 73.2

Example 5 Evaluation of Binding Affinity of Bispecific scFv2Fc Proteinsto Human 5T4 and Human CD137 Methods

Expression and purification of recombinant human 5T4 and human CD137Extracellular Domains (ECDs): Using standard molecular biologytechniques and starting with a vector encoding the full length sequenceof the 5T4 and CD137 proteins, nucleotide primers were designed toamplify the extracellular regions of human 5T4 and human CD137. Anadditional peptide sequence was added to the c-terminus of ECD at theposition where the native protein would be predicted to enter themembrane. This peptide contained recognition sequences that could beutilized for affinity purification purposes. The expression vectors weretransiently transfected into HEK-293 cells. The recombinant ECD waspurified from the conditioned media using immobilized metal affinitychromatography (IMAC) and size exclusion chromatography. Protein puritywas verified using analytic SEC.

Surface Plasmon Resonance (SPR) affinity analyses of bispecific proteinsto recombinant CD137 and 5T4 ecto-domain: SPR binding affinity studiesof bispecific anti-5T4×anti-CD137 proteins to recombinant monomeric 5T4and CD137 ectodomain (ECD) were conducted at 25° C. in HBS-EP⁺ buffer ona Biacore T200 system. Goat anti-human IgG F(ab′)2 fragment (JacksonImmunoResearch) at 20 μg/mL in 10 mM sodium acetate (pH 4.5) wasimmobilized at a density of 2000 response units (RU) onto the flow cellof a CM5 research-grade sensor chip (GE) by standard amine couplingchemistry. Each bispecific variant at 200 nM in HBS-EP+ buffer wascaptured in the flow cell with the immobilized anti-human IgG at a flowrate of 50 μL/min for 60 sec to reach ˜500 RU response, leaving one flowcell surface unmodified as the reference. Following a 30 secstabilization period, four different concentrations of each ECD (0, 20,60, and 180 nM) were injected at 50 μL/min for either 120 sec followedby a 240 sec dissociation period (for 5T4) or 90 sec followed by a 180sec dissociation period (for 4-1BB). Regeneration was achieved byduplicate injections of 10 mM glycine (pH 1.5) at a flow rate of 50μL/min for 30 sec followed by HBS-EP+ buffer stabilization for 1 min.

Sensorgrams obtained from kinetic SPR measurements were analyzed by thedouble subtraction method. The signal from the reference flow cell wassubtracted from the analyte binding response obtained from flow cellswith immobilized or captured ligands. Buffer reference responses werethen averaged from multiple injections. The averaged buffer referenceresponses were then subtracted from analyte binding responses, and thefinal double-referenced data were analyzed with Biacore T200 Evaluationsoftware (2.0, GE), globally fitting data to derive kinetic parameters.All sensorgrams were fitted using a simple one-to-one binding model.

Results

Table 19 shows the binding parameters determined by SPR for ADAPTIR™bispecifics that bind to CD137 and 5T4. It should be noted thatperforming affinity measurements in this format, with the bispecificmolecule captured on the chip and injecting recombinant monomeric CD137or 5T4 ECD should result in true affinity values that are not confoundedby avidity effects. As Table 19 illustrates, it was possible via carefulscreening of the randomized phage libraries to isolate binding domainvariants that led to significant improvement in binding affinity eitheranti-CD137 and/or anti-5T4 scFv. A large reduction in the Hu CD137 KDvalue was achieved with the optimized ADAPTIR™ bispecifics ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-209, and ALG.APV-210 in comparison tothe unoptimized parent construct ALG.APV-006. Similarly, ALG.APV-179,ALG.APV-187, ALG.APV-209, and ALG.APV-210 exhibited improved affinityvalues for human 5T4 when compared to the unoptimized ALG.APV-006.

TABLE 19 Binding affinity (K_(D)), disassociation, and associationconstants for bispecific molecules binding to recombinant human CD137 orhuman 5T4 Affinity to Hu CD137 Affinity to Hu 5T4 by SPR by SPR KD k_(a)k_(d) KD k_(a) k_(d) Construct (nM) (1/Ms) (1/s) (nM) (1/Ms) (1/s)ALG.APV-006 210 2.8E+05 5.9E−02 109 2.4E+04 2.6E−03 ALG.APV-178  703.9E+05 2.7E−02 106 2.3E+04 2.5E−03 ALG.APV-179  71 3.9E+05 2.8E−02  292.2E+04 6.2E−04 ALG.APV-187  73 3.7E+05 2.7E−02  35 2.1E+04 7.2E−04ALG.APV-209  59 5.6E+05 3.3E−02  66 1.4E+04 9.4E−04 ALG.APV-210  595.5E+05 3.3E−02  79 1.5E+04 1.2E−03

Example 6 Evaluation of Thermal Stability of Bispecific scFv-Fc-scFvProteins to Human 5T4 and Human CD137

Differential Scanning calorimetry (DSC) and Differential ScanningFluorimetry (DSF) are tools typically employed to measure assess thestructural stability of recombinant proteins. By measuring the energyrequired to increase the temperature of a protein sample, it is possibleto determine the midpoint temperature of melting (Tm) of individualprotein domains. It is generally accepted that protein domains withhigher Tm values are considered to be more stable. DSF analysis wasperformed with a set of optimized bispecific ADAPTIR™ bispecificproteins (ALG.APV-178, ALG.APV-179 and ALG.APV-187) and compared to theunoptimized ADAPTIR™ bispecific version (ALG.APV-006) (Table 20). Asignificant increase in Tm of the 1618 anti-CD137 and 1210 anti-5T4 wasobserved after incorporation of beneficial mutations that wereidentified by a random mutagenesis phage display approach describedabove.

TABLE 20 Midpoint of melting temperature, Tm, of optimized ADAPTIR ™bispecifics versus unoptimized constructs Tm, Anti-CD137 Tm, Anti-5T4Optimized? Construct scFv, ° C. scFv, ° C. N ALG.APV-006 56.5 70.2 YALG.APV-178 60.7 73.0 Y ALG.APV-179 61.1 73.0 Y ALG.APV-187 61.1 73.5

Example 7 Binding of Bispecific scFv-Fc-scFv Proteins Molecules to Humanand Non-Human Primate CD137(+) Cell Lines

To confirm that binding to CD137 on the surface of cells was not lostupon scFv conversion of the variable domains or the mutationsintroduced, flow cytometry was used to quantitate binding of constructedbi-specific CD137×5T4 binding ADAPTIR™ molecules to cell linesexpressing human or cynomolgus macaque CD137.

Binding studies on CHO-K1 cells lines expressing CD137 were performed bystandard flow cytometry-based staining procedures. CHO-K1 cells(generated by Alligator) were transfected with human or cynomolgusmacaque CD137. A typical experiment would label approximately 100,000cells per well, in 96-well plates, with a range of 100 nM to 0.05 nMbinding molecule in 50 μL of saline buffer with 2% BSA and 2 mM EDTA,for 30 min on ice, followed by washes and incubation with PE-labeledsecondary antibody, goat anti-human IgG Fcγ (Jackson Laboratory), for 20minutes on ice. Signal from bound molecules was detected using a LSR-II™or a Symphony A3 flow cytometer (BD Biosciences) and analyzed by FlowJoflow cytometry analysis software. Median fluorescence intensity(MFI-median) of bound molecules on live cells was determined afterexclusion of doublets cells. Nonlinear regression analysis to determineEC₅₀ values was performed in GraphPad Prism 6® graphing and statisticssoftware.

FIG. 2 shows the binding curves of seven ADAPTIR™ molecules(ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191,ALG.APV-196 and ALG.APV-198) to the CHO-K1/human CD137 cell line. Allthe molecules showed similar levels of binding (EC₅₀ values) andsaturation. FIG. 3 shows the binding curves of the same seven constructsto the CHO-K1/cynomolgus CD137 cell line. Similar levels of binding andsaturation were observed by all seven constructs on the cynomolgustarget. FIG. 12 shows the binding curves of six ADAPTIR™ molecules(ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209, andALG.APV-210) and the Morrison format control ALG.APV-004 to theCHO-K1/human CD137 cell line. All the molecules bound with similar EC₅₀values in the range of 0.3 to 0.6 nM, and similar levels of saturation.

Example 8 Binding of Bispecific scFv-Fc-scFv Proteins to Human andNon-Human Primate 5T4(+) Cell Lines

To compare the binding of the 5T4 on the surface of cancer cells afterchanges to the scFv domains were introduced, flow cytometry was used toquantitate binding of constructed bi-specific CD137×5T4 binding ADAPTIR™molecules to cell lines expressing 5T4.

Binding of bispecific CD137×5T4 molecules to 5T4(+) cell lines: Bindingcharacteristics of the bispecific ADAPTIR™ molecules (ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-196, ALG.APV-208, ALG.APV-209 andALG.APV-210) were compared. Binding studies were performed by standardflow cytometry-based staining procedures two cell lines expressing5T4:CHO-K1 cells (obtained from Alligator) transduced with human orcynomolgus macaque 5T4 and SKOV-3 human ovarian adenocarcinoma cellswhich express (human) 5T4. All labeling and washes were performed inU-bottom 96-well plates in saline buffer with 0.1% BSA and 2 mM EDTA.Cell were plated at approximately 100,000 cells per well and incubatedwith a range of 100 nM to 0.05 nM concentrations of test molecules in 50μL volume/well, for 30 minutes on ice. Cells were washed three timesthen incubated for another 30 min on ice with fluorescently-labeledsecondary polyclonal antibody, F(ab′)² goat anti-human IgG (JacksonImmunoResearch Laboratories). The cells were then washed twice and thesamples acquired in a BD LSRII or a Symphony A3 flow cytometer. Thesample files were analyzed using FlowJo software; the mean fluorescenceintensity (MFI-mean) or median fluorescence intensity (MFI-median) ofthe live population of cells in each well was calculated after gating onlive cells (forward vs side scatter). Nonlinear regression analysis todetermine EC₅₀ values was performed in GraphPad Prism 6® graphing andstatistics software.

FIG. 4 shows the binding curves of seven ADAPTIR™ molecules(ALG.APV-006, ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-191,ALG.APV-196, and ALG.APV-198) to the CHO-K1/human 5T4 cell line. Theconstruct ALG.APV-006 bound with an EC₅₀ value >10 nM, while theremaining ADAPTIR™ constructs (ALG.APV-178, ALG.APV-179, ALG.APV-187,ALG.APV-191, ALG.APV-196, and ALG.APV-198) bound with EC₅₀ values in therange of 3 to 10 nM. The differences in EC₅₀ values between constructswere more evident when binding assays were conducted on SKOV-3 cells.SKOV-3 cells express approximately 10-fold lower levels of human 5T4than the CHO-K1/human 5T4 transfectants (FIG. 5). FIG. 6 shows thebinding curves of the ALG.APV-006, ALG.APV-178, ALG.APV-179,ALG.APV-187, ALG.APV-196, and ALG.APV-198 of constructs on SKOV-3 cells.The constructs ALG.APV-006, ALG.APV-078, ALG.APV-179, and ALG.APV-187bound with slightly lower affinity (higher EC₅₀ values) and highermaximum binding levels than the ALG.APV-196 and ALG.APV-198 constructs.FIG. 7 shows the binding curves of ALG.APV-006, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196, and ALG.APV-198 onthe CHO-K1/cynomolgus 5T4 transfectants. Similar to the binding profileon the human 5T4(+) cells, constructs ALG.APV-006, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-191, ALG.APV-196, and ALG.APV-198displayed lower affinity and higher maximum binding levels thanconstructs ALG.APV-196 and ALG.APV-198.

FIG. 13 shows the binding curves of six ADAPTIR™ molecules (ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210) andthe Morrison format control ALG.APV-004 to murine CT26 cells (ATCC)expressing human 5T4. These cells express levels of human 5T4 comparableto those observed on SKOV-3 cells (FIG. 5). All the constructs boundwith EC₅₀ values in the range of 1 to 3 nM. Constructs ALG.APV-179,ALG.APV-187, ALG.APV-209, and ALG.APV-210 showed higher levels ofmaximum binding than ALG.APV-178 and ALG.APV-208. The Morrison formatcontrol molecule ALG.APV-004 showed the lowest levels of maximumbinding. FIG. 14 shows the binding curves of ALG.APV-004, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209, and ALG.APV-210 onthe CHO-K1/cynomolgus 5T4 transfectants. All the molecules bound withsimilar EC₅₀ values, in the range of 6 to 10 nM, and similar levels ofsaturation. FIG. 15 shows the binding curves of ALG.APV-004,ALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 andALG.APV-210 on a human cell line expressing neither CD137 nor 5T4(MOLM13 cells, ATCC). None of the molecules showed binding at anyconcentration.

Example 9 Binding of a 5T4-CD137 Bispecific Antibody to 5T4 ExpressingCell Lines

Experiments were performed to determine the binding of ALG.APV-210 tohuman 5T4 expressed on cells at different receptor densities. Both humantumor cell lines with endogenous levels of 5T4 and cells transfected toproduce 5T4 were assessed for target binding of ALG.APV-210.

Materials and Methods

Biotinylation: In order to detect ALG.APV-210 binding to 5T4-expressingcell lines, ALG.APV-210 was first biotinylated. The quality of thebiotinylated protein and the impact on binding to its targets wasassessed using ELISA.

FACS staining and analysis of 5T4-expressing cell lines: Prior to theFACS staining, all cell lines were characterized for their 5T4expression levels using a receptor density kit (Quantum Simply Cellular,anti-human IgG, Bangs Laboratories, Inc). Information on the cell linesused and their respective 5T4 expression densities are shown in Table21. Adherent cell lines were cultured according to their supplier'sinstructions and were harvested from their cell culture flasks usingTrypsin/EDTA, counted in R10 medium in a Burker chamber, and diluted inFACS buffer (PBS+0.5% BSA, 0.05% NaN₃) to a concentration of 4×10⁶cells/mL. 50 μL (0.20×10⁶) of cell solution was added to a 96-well FACSstaining plate (Falcon #351190). Cells were incubated for 20 min in 4°C. with 50 μL of the biotinylated ALG.APV-210 (2× final concentration).The antibodies were added in a serial dilution starting at 20 μg/mLtitrating down in a 12 step 1/2 dilution down to 0.010 μg/mL. Following2× washing with FACS buffer, cells were incubated for 30 min in 4° C.with 100 μL of a secondary antibody (streptavidin-PE, BD #554061,diluted 1/500). Following 2× washing steps with FACS buffer, cells werere-suspended in 200 μL of cell fixation (BD CellFIX 10× BD, #340181,diluted in MQ H2O). Cells were analysed for ALG.APV-210 binding usingflow cytometry (FACSVerse) by gating on single cells. MFI values(secondary antibody only subtracted) from 1 out of 2 independentexperiments, as well as normalized data and EC₅₀-values of pooled datafrom 2 experiments were determined and plotted using GraphPad Prism 7,non-linear regression log (agonist) vs. normalized response-variableslope.

TABLE 21 Cell lines expressing 5T4 and receptor densities/cell 5T4 EC₅₀95% Cells Cancer type density/cell (nM) CI HCT-116 Human colon carcinoma62,000 16 15-17 HT-29 Human colon carcinoma 30,000 12 11-14 JAR Humanchoriocarcinomas 69,000 9  5-17 JEG Human choriocarcinomas 66,000 8 5-12 SKOV-3 Human ovarian cancer 60,000 15 12-18 BxPC-3 Pancreas cancer150,000 20 18-22 B16-hu5T4 Transfected mouse 320,000 14  9-19 melanomaCHO-hu5T4 Transfected 4,100,000 23 18-28 CT26-hu5T4^(high) Transfectedmouse colon 1,000,000 20 16-27 carcinoma CT26-hu5T4^(int) Transfectedmouse colon 200,000 12 11-13 carcinoma CT26-hu5T4^(low) Transfectedmouse colon 50,000 11  8-15 carcinoma

Results and Conclusions

Using ELISA, the binding of biotinylated ALG.APV-210 to 5T4 wasdetermined to be similar to that of the non-biotinylated ALG.APV-210(data not shown).

The binding (EC₅₀) of the biotinylated ALG.APV-210 to 5T4 when expressedon cells, either at endogenous levels on human tumor cells or on cellstransfected to express 5T4 was assessed. As shown in FIG. 39 using MFI,ALG.APV-210 binds to both human tumor cells expressing endogenous levelsof 5T4 and transfected cell lines in a dose-dependent manner.

Table 21 above shows the binding EC₅₀ of ALG.APV-210 to 5T4 positivehuman tumor cell line and to 5T4 transfected cells determined fromnormalized MFI values (background subtracted) from 2 pooled experimentsplotted in a dose-response curve shown in FIG. 40. The mean EC₅₀ valuesranged between 8-23 nM for the different 5T4 positive cell lines. Inconclusion, ALG.APV-210 binds to 5T4-expressing cells at an EC₅₀ rangingbetween 8-23 nM.

Example 10 Agonistic Function of Bispecific scFv-Fc-scFv Proteins inReporter Assays

To compare the effectiveness of different bispecific CD137-bindingmolecules at inducing target-dependent activation of CD137, sevendifferent bispecific anti-CD137×anti-5T4 molecules were compared in aCD137 reporter assay, as described in Example 3.

CD137 Reporter Assay

Jurkat/CD137 transfectants carrying a luciferase reporter gene under thecontrol of an NF-κB promoter (Promega) were cultured according to themanufacturer's protocols. Jurkat/NF-κB reporter cells were cultured withhuman primary ductal breast carcinoma cells HCC1143 (ATCC), whichexpress human 5T4, at approximately 15,000 reporter cells to 30,000target cells in 96-well plates. Concentrations of bispecific moleculeswith final concentration ranging from 10 nM to 0.002 nM were added.Cells were cultured in a total volume of 100 μL of RPMI 1640 mediasupplemented with 10% fetal bovine serum, sodium pyruvate, antibioticsand non-essential amino acids. Plates were incubated at 37° C., 5% CO₂in humidified incubators for 5 to 6 hours. One hundred microliters ofBio-Glow buffer (Promega) was added to each well and incubated for 5 to10 minutes before measuring fluorescence. Luminescence was measured in aMicroBeta² 2450 Microplate Counter (Perkin Elmer). Nonlinear regressionanalysis to determine EC₅₀ values was performed in GraphPad Prism 6®graphing and statistics software.

Results

In FIG. 8, all the constructs display agonistic function in the presenceof 5T4(+) cells; no reporter activity was observed in the absence of5T4(+) cells (data not shown). The constructs ALG.APV-178, ALG.APV-179,ALG.APV-187, ALG.APV-191, ALG.APV-196 and ALG.APV-198 displayed betteragonist function (up to 6-fold lower EC₅₀ values) than the non-optimizedADAPTIR™ construct, ALG.APV-006, and the Morrison format control,ALG.APV-004. Every point in the curve represents the average ofduplicate wells. The y-axis shows values in relative fluorescence units(RLU). FIG. 16 shows the activity of the bispecific constructs inJurkat/NF-κB reporter cells, after 5 hours of incubation in the presenceof 5T4(+) cells (HCC1143) or 5T4(−) cells (MOLM13). The constructsALG.APV-178, ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 andALG.APV-210 displayed better agonist function (10-fold lower EC₅₀values) than the Morrison format control, ALG.APV-004. No reporteractivity was observed in the presence of 5T4(−) cells (MOLM13). Everypoint in the curve represents the average of duplicate wells. The y-axisshows values in relative fluorescence units (RLU).

Example 11 Agonistic Function of Bispecific scFv-Fc-scFv Proteins inPrimary T Cell Assays

The agonistic function of different anti-CD137×anti-5T4 bispecificmolecules at inducing target-dependent activation of CD137 was tested incultures of peripheral blood mononuclear cells (PBMC). In order to testthe function of the anti-CD137 bispecific constructs, primary PMBC werestimulated with anti-CD3 antibodies to upregulate CD137, which is notexpressed on resting T cells. Co-stimulation of primary T cells throughthe TCR and CD137 enhances T-cell secretion of the cytokine interferongamma (IFN-γ).

CD137 Stimulation of PBMC:

Peripheral blood mononuclear cells (PBMC) were isolated from human bloodusing standard ficoll gradients. The isolated cells were washed insaline buffer. PBMC were cultured with CHO-K1/human 5T4 cells, atapproximately 120,000 PBMC to 30,000 5T4 (+) cells in 96-well plates.Anti-CD3 antibody OKT3 (eBioscience) was added to all the wells at aconcentration of 0.1 μg/mL. Cells were cultured in a total volume of 200μl of RPMI 1640 media supplemented with 1% fetal bovine serum, sodiumpyruvate, antibiotics and non-essential amino acids. Plates wereincubated at 37° C., 5% CO₂ in humidified incubators for 72 hours. Onehundred microliters of media were collected at 72 hours. Levels of theIFN-γ were measured in the supernatant using Milliplex® kits (Millipore)using the manufacturer's instructions. Data was collected in a Bio-PlexReader 200 System (Bio-Rad). Nonlinear regression analysis to determineEC₅₀ values was performed in GraphPad Prism 6® graphing and statisticssoftware.

FIG. 9 shows the levels of IFN-γ induced by ALG.APV-006, ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-196, ALG.APV-198 and the Morrisonformat control ALG.APV-004. All the constructs induced higher levels ofIFN-γ than anti-CD3 alone, in a dose-dependent manner. Every point inthe curve represents the average of duplicate wells. FIG. 17 shows thelevels of IFN-γ induced in primary PBMC cultures at 72 by ALG.APV-178,ALG.APV-179, ALG.APV-187, ALG.APV-208, ALG.APV-209 and ALG.APV-210 andthe Morrison format control construct ALG.APV-004 in the presence ofCHO-K1/human 5T4 cells. All the constructs induced higher levels ofIFN-γ than anti-CD3 alone, in a dose-dependent manner. Every point inthe curve represents the average of duplicate wells.

The functional activity of anti-5T4×anti-CD137 bispecific constructs wasalso evaluated in a CD8+ T cell assay wherein cells were cultured inmicrotiter plates coated with a 5T4-Fc construct and an anti-CD3antibody.

5T4 Stimulation of Purified T Cells:

PBMCs were isolated by density gradient centrifugation usingFicoll-Paque (p 1.077 g/mL) (GE Healthcare #17-1440-02) from leukocyteconcentrates obtained from healthy donors (Clinical Immunology andTransfusion Medicine, Labmedicin Region Skåne, Lund Sweden). CD8+ Tcells were enriched by negative selection using the CD8+ T cellisolation kit (Miltenyi 130-096-495). Plates were coated overnight at 4°C. with 3 μg/mL α-CD3, clone OKT3 (Affymetrix eBioscience #16-0037-85),washed and coated with 5 μg/mL 5T4-Fc for 2 h at 37° C. After coatingwith 5T4-Fc, plates were washed and blocked for a minimum of 30 minuteswith RPMI (Gibco #61870010) containing 10% FCS (Heat inactivated, Gibco#10270-106 lot 41Q9248K) and 10 mM Hepes (Gibco #15630056). Bispecificconstructs were diluted in RPMI containing 10% FCS and 10 mM Hepes andadded to the plates 30 minutes before addition of CD8+ T cells (0.07×10⁶cells/well). Assay plates were incubated for 72 h at 37° C., after whichculture supernatants were harvested. IFN-γ levels in the supernatantswere measured by ELISA (BD OptiEIA #555142). The experiment was repeatedtwice with a total of 3 donors. Results from the first experiment areshown in FIG. 11A. Results of control samples, wherein cells andbispecific constructs were cultured in plates that were not coated with5T4-Fc, are shown in FIG. 11B.

FIG. 11A shows that the ADAPTIR™ format anti-5T4×anti-CD137 bispecificconstructs (ALG.APV-178, ALG.APV-179, ALG.APV-196, and ALG.APV-198) haveincreased functional activity compared to the Morrison format bispecificconstructs (ALG.APV-004), demonstrated by an increase in the IFN-γlevels present in the culture supernatants in the presence of 5T4. Infact, the ADAPTIR™ format constructs induced IFN-γ levels that were2-4.5 times higher than those induces by the Morrison format constructs(FIG. 11A).

Example 12 Antigen Dependent Localization of Bispecific Antibodies in5T4 Positive Tumors In Vivo

Antigen-dependent localization of the bispecific constructs targeting5T4 and 4-1BB to 5T4-positive B16-5T4 tumors was evaluated in wild typeC57BL/6 mice.

Materials and Methods

Mice: 8 week old, female C57BL/6 mice from Janvier, France were used forthe experiments. All experiments were done by approval of the Malmö/Lundethical committee.

Bispecific constucts: Two optimized 5T4- and CD137-targeting bispecificconstructs in the ADAPTIR™ format, ALG.APV-209 and ALG.APV-210, wereused. ALG.APV-004, a bispecific construct in the Morrison format, servedas a positive control. Vehicle administration served as a negativecontrol.

Cells: B16.F10 WT (B16) cells were obtained from ATCC and cultivatedaccording to their recommendations. B16 cells expressing human 5T4(hereafter B16-5T4) were obtained from Professor Peter Stern and grownunder 1.2 mg/mL of G418 selection medium.

Methods: For the antigen dependent localization of bispecificconstructs, a B16 melanoma twin tumor model was used, where each mousereceived one 5T4 negative and one 5T4 positive tumor at each side of thehind flank/back. The tumor cell lines, growing in log phase, wereinjected subcutaneously (1×10⁵ cells in 100 μL) on day 0.Intraperitoneal construct treatments (100 μg) were given on day 6 and 13and mice were sacrificed on day 14 (24 h after the final treatment).

FACS Analysis: Tumors for flow cytometry analysis were mechanically andenzymatically digested using Liberase TL and DNase I (Roche) and passedthrough a 70 μm strainer. The resulting single cell suspension wasstained for FACS analysis. Briefly, non-specific binding of theconstructs was blocked using mouse IgG or Fc block. Dead cells wereexcluded using Fixable Viability Stain 450 (Molecular probes) accordingto manufacturer's instructions. Binding of the two ADAPTIR™ bispecificconstructs or the control constructs to the tumor cells was analysedusing a secondary antibody: goat-anti-human Fc-PE (JacksonImmunoresearch). Alternatively, detection of the bispecific constructswas performed using biotinylated 4-1BB antigen followed bystreptavidin-PE/PerCP-Cy5.5. Samples were analyzed on a BD FACSVerse anddata was analysed using FlowJo software. Statistical analysis wasperformed using Mann-Whitney non-parametric T-test, 2-tail and GraphPadprism program.

Immunohistochemistry: Tumors for immunohistochemistry were snap frozenin isopropanol on dry ice. Cryosections (8 mm) were stained for 5T4expression using a rabbit anti-human 5T4 (Abcam) or rabbit anti-human Fc(Jackson Immunoresearch) followed by anti-rabbit Brightvision-HRP(Immunologic) and DAPI staining. The immunohistochemical staining wasassessed as follows; negative (0), weak staining (1+), moderate staining(2+), or strong staining (3+).

Results

Both bispecific constructs in the ADAPTIR™ format, ALG.APV-209 andALG.APV-210, and the positive control, ALG.APV-004, localized to5T4-expressing B16 tumors, but not to 5T4-negative tumors (B16.F10).This was demonstrated both by flow cytometry (FIGS. 18A and 18B) andimmunohistochemistry (FIGS. 19A and 19B).

Both the bispecific constructs (ALG.APV-209, ALG.APV-210) and thepositive control (ALG.APV-004) demonstrated statistically significant5T4-dependent localization to antigen-positive tumors compared to thenegative vehicle control as measured by flow cytometry (FIG. 18A). Thebinding to 5T4-expressing tumors could also be detected usingbiotinylated 4-1BB for detection (FIG. 18B), confirming that thebispecific molecules were intact.

The localization was further demonstrated by immunohistochemistry (FIGS.19A and 19B and Table 22). The bispecific antibodies bound strongly to5T4 positive tumors (B16-5T4, FIG. 19A) but not to the 5T4 negativetumors (B16.F10, FIG. 19B).

TABLE 22 In vivo localization of bispecific constructsand vehiclecontrols Treatment 5T4-negative tumors 5T4-positive tumors ALG.APV-209 −+++ ALG.APV-210 − +++ ALG.APV-004 − +++ Vehicle − −

Example 13 In Vitro Activity of a 5T4-CD137 Bispecific Antibody in anIFNγ Release Assay Using Human CD8 T Cells and 5T4-CT26 Tumor Cells

The functional activity of the 5T4-CD137 bispecific antibody ALG.APV-210was evaluated in a CD8 T cell assay, where CD8 T cells, stimulated withCD3 antibodies coated on beads, were co-cultured in plates with CT26tumor cells expressing different levels of 5T4.

CT26 tumor cells, a murine colon carcinoma cell line (ATCC® CRL-2638)was previously transfected to express human 5T4 and then single cellsorted using flow cytometry to generate single cell clones expressingeither high or low levels of human 5T4 (for receptor density, see Table23). 5T4-CT26 tumor cells or non-transfected wildtype cells (CT26 wt)were UV irradiated using a UVB crosslinker (AnalytikJena, Lamp: 254 nm).After irradiation of the plate, the cells were washed once in medium andthen resuspended at a concentration of 4×10⁶ cells/ml in new growthmedium R10 (RPMI, Gibco #61870010 containing 10% heat inactivated FBS,Hyclone #SV30160 lot RB35944 and 10 mM Hepes, Gibco #15630056). 50 μL(2×10⁵ cells/well) of the UV-irradiated CT26-h5T4^(high),CT26-h5T4^(low) or CT26 wt cells were added to TC treated assay plates(Eppendorf #0030 730.119) and incubated in 37° C. overnight.

TABLE 23 5T4 receptor density on human 5T4 transfected and single cellsorted CT26 cells CT26 cell clone 5T4 receptor density/cellCT26-h5T4^(high) 1 × 10⁶ ± 2 × 10⁵ CT26-h5T4^(low) 5 × 10⁴ ± 1 × 10⁴CT26 wt 0

The following day after UV irradiation of the CT26 cells, human PBMCwere isolated by density gradient centrifugation using Ficoll-Paque (GEHealthcare #17-1440-02) from leucocyte concentrates obtained fromhealthy donors (Clinical Immunology and Transfusion Medicine, LabmedicinRegion Skåne, Lund Sweden). CD8+ cells were enriched by negativeselection using the CD8+ T cell isolation kit (Miltenyi 130-096-495).

The bispecific antibody ALG.APV-210 was diluted in a serial dilution inR10 medium and 50 μL was added to each well of the assay plates andincubated at 37° C. for 30 min, prior to the addition of 50 μL of αCD3beads (4×10⁸/mL of anti-CD3 antibodies clone: OKT-3, AffymetrixeBioscience #16-0037-85, coated on beads, diluted to 2×10⁶/mL) at a 1:1CD8+T cell to beads ratio, followed by the addition of 50 μL of theeffector cells (enriched CD8+ cells, 2×10⁶/mL or 1×10⁵/well). The assayplates were incubated for 72 h at 37° C., and culture supernatantharvested. IFNγ levels in the supernatants were measured by ELISA (BDOptiEIA #555142). EC50 was determined by non-linear regression log(agonist) vs. Normalized response (variable slope) using GraphPad Prism7. TOP value (maximal functional effect) was determined by calculatingthe mean of the two highest values of IFNγ production in the culturesupernatant. The experiment was performed 5 times, including in total 12donors.

The study provided the functional effect of the CD137-5T4 bispecificantibody ALG.APV-210 using human CD8 T cells (the effector cells)co-cultured in plates with CT26 cells expressing different levels of 5T4(for crosslinking of CD137 via binding to 5T4). As shown in FIG. 20, thebispecific CD137-5T4 antibody ALG.APV-210 induces a potent T cellactivation, measured by IFNγ release, in a dose-dependent manner in thepresence of CT26 cells expressing either high (CT26-h5T4^(high)) or low(CT26-h5T4^(low)) levels of 5T4. IFNγ release is presented in FIG. 20 asnormalized data of ALG.APV-210;mean values of 12 donors from 5 pooledexperiments are shown.

Table 24 shows the EC₅₀ of ALG.APV-210 in the CD8 T cell assay in thepresence of human 5T4 expressing CT26 cells. Log (agonist) vs.Normalized response (variable slope) mean values and 95% CI of 12 donorsfrom 5 pooled experiments is shown in the Table. As shown in FIGS. 21Aand 21B, the maximal functional effect of ALG.APV-210 was dependent onthe expression level of 5T4 on the CT26 cells, as illustrated by aslightly lower secretion of maximal IFNγ release in co-culture withCT26-h5T4^(low) cells compared to co-cultures with CT26-h5T4^(high)cells (the maximal effect of CT26-h5T4^(low) was 82% ofCT26-h5T4^(high)). In the absence of 5T4-induced crosslinking usingCT26-wt cells there is no or very low T cell activity (7% of maximaleffect of CT26-h5T4^(high)). Max IFNγ release is presented in FIG. 21 asabsolute values (ng/mL) (FIG. 21A) or normalized values relative toCT26-h5T4^(high) (FIG. 21B). Mean and SEM values of 12 donors from 5pooled experiments are shown.

TABLE 24 Binding (EC₅₀) of ALG.APV-210 in the presence of human 5T4+CT26 cells ALG.APV-210-h5T4^(high) ALG.APV-210-h5T4^(low) EC₅₀ (nM) 0.410.24 95% CI 0.36 to 0.46 0.20 to 0.28

Example 14 Binding of a 5T4-CD137 Bispecific Antibody to CD137 on Humanand Cynomolgus Primary CD3-Stimulated CD8 T Cells

A study was conducted to compare the relative binding of ALG.APV-210towards human and cynomolgus CD137 expressed on activated primary CD8 Tcells (gated from CD3-stimulated PBMC).

Materials and Methods

In order to detect ALG.APV-210 binding to primary human and cynomolguscells in FACS, ALG.APV-210 was first biotinylated. The quality of thebiotinylated protein was assessed using HPLC and the impact on bindingto its targets was assessed using ELISA. ANC017, an antibody in theADAPTIR format with the same framework as ALG.APV-210 (germline CDRs)was used as a negative control and was also biotinylated.

Isolation and stimulation of human PBMC: Human PBMC were isolated bydensity gradient centrifugation using Ficoll-Paque (GE Healthcare#17-1440-02) from leucocyte concentrates obtained from 3 human healthydonors (Clinical Immunology and Transfusion Medicine, Labmedicin RegionSkåne, Lund Sweden). Non-tissue treated 96 well plates (Nunc #268200)were pre-coated overnight at 4° C. with 10 μg/mL αCD3, clone OKT3(Affymetrix eBioscience #16-0037-85). The following day, plates werewashed in PBS, and human PBMC diluted in R10 (RPMI, Gibco #61870010containing 10% heat inactivated FBS, Hyclone #SV30160 lot RB35944 and 10mM Hepes, Gibco #15630056), were added at a concentration of 0.2×10⁶cells/well and incubated in 37° C. in wells with or without CD3stimulation for 48 h.

Isolation and stimulation of cynomolgus PBMC: Cynomolgus PBMC wereisolated from 20 mL of cynomolgus monkey blood from 3 different donors,obtained from Silabe, France by density gradient centrifugation usingLympholyte-mammal (Cedarlane labs) according to manufacturer'sinstructions. Non-tissue treated 96 well plates were pre-coatedovernight at 4° C. with 3 μg/mL α-monkey CD3, clone FN-18 (Invitrogen#APS0301). The cynomolgus PBMC were then stimulated identically as thehuman PBMC described above

FACS staining and analysis of CD8 T cells: Following 48 h of incubationwith or without CD3, human and cynomolgus PBMC were harvested, pooled,recounted and diluted in FACS buffer (PBS+0.5% BSA, 0.05% NaN₃) to aconcentration of 5×10⁶ cells/mL. 50 μL (0.25×10⁶ cells) of cell solutionwas added to a 96-well FACS staining plate (Falcon #351190). Following10 min of Fc-blocking in RT using Beriglobin (hIgG, 200 μg/mL), cellswere washed in FACS buffer and then incubated for 1 h in 4° C. with 100μL of the biotinylated ALG.APV-210 or negative control. The antibodieswere added in a serial dilution starting either at 5 μg/mL titratingdown in a 12 step 1/3 dilution down to 0.0003 μg/mL (exp 1) or 1 μg/mLtitrating down in a 12 step 1/2 dilution down to 0.0005 μg/mL (exp 2).Following 2× washing with FACS buffer, cells were incubated for 30 minin 4° C. with 50 μL of a secondary antibody (streptavidin-APC, BD#554067) and 50 μL of fluorescent conjugated antibodies againstdifferent T cell surface markers (CD4-FITC #550628, CD3-PECy7 #557749and CD8-APC-H7 #5601797, BD). Following washing in PBS, cells werestained in 15 min in 4° C. with 50 μL of fixable viability stain BV510(in order to gate away non-viable cells), washed again and thenre-suspended in 130 μL of cell fixation (BD CellFIX 10× BD, #340181,diluted in MQ H2O). Cells were analysed for ALG.APV-210 binding usingflow cytometry by gating on viable, single cells expressing CD3 and CD8.MFI values (FMO subtracted) from 2 independent experiments as well asnormalized pooled data from 2 experiments and EC₅₀-values weredetermined and plotted using GraphPad Prism 7, non-linear regression log(agonist) vs. normalized response-variable slope, n=6 donors/group.

Results

In ELISA tests, the binding of biotinylated ALG.APV-210 to CD137 wassimilar to that of the non-biotinylated ALG.APV-210 (0.6 nM vs. 0.7 nM).The binding (EC₅₀) of the biotinylated ALG.APV-210 to itsimmunomodulatory target CD137 when expressed on activated primary humanand cynomolgus CD8 T cells from CD3 stimulated PBMC was assessed andcompared between the two species as well as to unstimulated cells. Asshown using MFI of all CD3+CD8+ cells in FIGS. 22A, 22B, and 22C, andTable 25, ALG.APV-210 binds to both human and cynomolgus CD8 T cells ina dose-dependent manner, but not to unstimulated cells. Binding to thenegative control, ANC107, was low/undetectable for CD3-stimulated orunstimulated human PBMC or cynomolgus PBMC cells. The maximum amount(MFI) of ALG.APV-210 bound to CD3-stimulated cynomolgus PBMC was higherin comparison to human PBMC, most likely due to a higher proportion ofthe CD3-stimulated cynomolgus PBMC expressing CD137, or due to agenerally higher expression of CD137 on the cynomolgus CD8 T cells. Itis uncertain whether the cynomolgus cells were more sensitive towardsCD3 stimulation in comparison to the human cells, and thereforeupregulated CD137 more, since the cells were stimulated with differentclones of anti-CD3 due to lack of cross-reactivity of the anti-CD3reagents between species.

TABLE 25 ALG.APV-210 binding (EC₅₀) to human and cynomolgus CD8 T cellsHuman CD8 T Cynomolgus CD8 T ALG.APV-210 binding cells (gated from cells(gated from (nM) CD3 stim PBMC) CD3 stim PBMC) EC₅₀ (nM) 0.235 0.236 95%CI (nM) 0.21 to 0.26 0.22 to 0.25

ALG.APV-210 binding was also assessed by looking at the percentage ofCD8 T cells bound by ALG.APV-210. There was a larger percentage ofcynomolgus CD8 T cells bound by ALG.APV-210 in comparison to the humanCD8 T cells (71-96% for cynomolgus, 65-76% for human, see FIGS. 23A,23B, and 23C). The binding EC₅₀ values were also calculated on the MFIof the CD137 positive cells, and the results was an EC₅₀ of 0.17 nM forbinding to human CD137 and 0.20 nM for cynomolgus monkey CD137.

Taken together, the results of the study showed that ALG.APV-210 bindswith a similar EC₅₀: 0.2 nM to CD3-stimulated primary CD8 T cells ofhuman and cynomolgus, but not to unstimulated cells.

Example 15 In Vitro Activity of a 5T4-CD137 Bispecific Antibody in anIFNγ Release Assay Using Human or Cynomolgus CD8 T Cells in the Presenceof 5T4

A study was conducted to compare the functional activity and determineEC₅₀ of ALG.APV-210 in an agonist assay using either human or cynomolgusmonkey CD8 T cells in the presence of plate immobilized 5T4.

Materials and Methods

CD8 T cells were suboptimally stimulated with plate immobilized CD3antibodies and activated by crosslinking of CD137 via ALG.APV-210binding to plate immobilized 5T4-Fc. CD8 T cell activation was measuredby determining IFNγ release in the cell culture supernatant.

Isolation and assay setup with human CD8 T cells: Human PBMC wereisolated by density gradient centrifugation using Ficoll-Paque (GEHealthcare #17-1440-02) from leucocyte concentrates obtained fromhealthy donors (Clinical Immunology and Transfusion Medicine, LabmedicinRegion Skåne, Lund Sweden). CD8+ cells were enriched by negativeselection using the CD8+ T cell isolation kit (Miltenyi #130-096-495).Non-tissue treated 96-well assay plates were coated overnight at 4° C.with 3 μg/mL anti-CD3 antibody (clone: OKT-3, Affymetrix eBioscience#16-0037-85). The following day, plates were washed in PBS and coatedwith 5 μg/mL 5T4-Fc for 2 h at 37° C. After 5T4-Fc immobilization, assayplates were washed in PBS and blocked for a minimum of 30 minutes withR10 (RPMI, Gibco #61870010 containing 10% heat inactivated FBS, Hyclone#SV30160 lot RB35944 and 10 mM Hepes, Gibco #15630056). ALG.APV-210 werediluted in a serial dilution in R10 and 50 μL was added in triplicatesto the assay plate 30 minutes prior to the addition of 50 μL of theeffector cells (enriched CD8 T cells, 1.4×10⁶ cells/mL or 0.07×10⁵cells/well). Assay plates were incubated for 72 h at 37° C., and culturesupernatant harvested. IFNγ levels in the supernatant was measured byELISA (BD OptiEIA #555142). The experiment was performed 4 times,including in total 8 donors.

Isolation and assay setup with cynomolgus CD8 T cells: Cynomolgus(Macaca fascicularis, adult males, 3-15 years) whole blood obtained fromSilabe, France was used. Red blood cells were removed by red blood celllysis (BD Pharma lyse, BD Biosciences, #555899) according to themanufactures protocol. CD8⁺ cells were enriched by positive selectionusing the CD8 MicroBead kit (Miltenyi Biotec, #130-091-112). Platescoated overnight at 4° C. with 1 μg/mL α-monkey CD3, clone FN-18(Invitrogen, #APS0301), washed and coated with 5 μg/mL 5T4-Fc for 2 h at37° C. After 5T4-Fc coating, plates were washed and blocked for aminimum of 30 minutes with R10. ALG.APV-210 was diluted in R10 and 50 μLwas added in triplicates to the plates 30 minutes before addition of 50μL of CD8 T cells (0.07×10⁶ cells/well). Assay plates were incubated for72 h at 37° C., and culture supernatant harvested. IFNγ levels in thesupernatants were measured by ELISA (Monkey IFNγ ELISA development kit,MABTECH, #3421M-1H-20). The experiment was performed 3 times, includingin total 8 donors. Obtained IFNγ levels from each human and cynomolgusdonor were normalized and means were calculated. The mean of thenormalized IFNγ levels from all 8 human or cynomolgus donors were pooledand the EC₅₀ was determined by non-linear regression (log agonist vsnormalized response, variable slope) using GraphPad 7.

Results

As shown in FIG. 24 and in Table 26, in the human CD8 T cell assay, thebispecific CD137-5T4 antibody ALG.APV-210 had a high functional activitywith an EC₅₀ of 0.2 nM. Normalized IFNγ levels from 4 experiments and atotal of 8 donors were pooled and the EC₅₀ determined by nonlinearregression using GraphPad 7. Mean and SEM are presented in FIG. 24.Absolute mean IFNγ values from two donors from one representativeexperiment is shown in FIGS. 25A and 25B. The top values of the anti-CD3reagents as well as background IFNγ levels varies from donor to donorwithin one experiment. Due to the observed variations between donors thedata was normalized prior to the analysis of the pooled data set. CD8 Tcell activation of ALG.APV-210 was assessed both in the presence andabsence of 5T4-Fc to verify a 5T4 crosslinking dependency. The resultsshowed that the bispecific CD137-5T4 antibody ALG.APV-210 induces apotent T cell activation, measured by IFNγ release, when cross-linked by5T4-; in the absence of 5T4 there is no T cell activity (FIGS. 25A and25B).

The functional effect of the CD137-5T4 bispecific antibody ALG.APV-210when using cynomolgus CD8 T cells was also assessed. Based on pooleddata from 3 experiments and a total of 8 donors, the EC₅₀ of ALG.APV-210in the cynomolgus CD8 T cell assay was determined to be 0.4 nM (FIG. 26and Table 26). Absolute mean values from 3 donors from onerepresentative experiment is also shown in FIGS. 27A, 27B, and 27C.Similar to the human setting, the absolute values (including top valuesand background IFNγ levels) also vary between individual cynomolgusdonors within one experiment. Therefore, the data was normalized priorto the EC₅₀ analysis of the pooled data set. CD8 T cell activation ofALG.APV-210 was assessed both in the presence and absence of 5T4-Fc, toverify a dependency of 5T4-induced crosslinking of CD137. As shown inFIGS. 27A-27C, the agonistic effect of ALG.APV-210 is 5T4-dependent inthe cynomolgus CD8 T cell assay in a similar manner as observed in thehuman setting. Each of FIGS. 27A, 27B, and 27C show absolute mean andSEM values of IFNγ of 3 individual donors from one representativeexperiment. As negative control, wells without (w/o) 5T4 were included.

TABLE 26 Binding of ALG.APV-210 in human and cynomolgus CD8 T cell assayHuman CD8 Cynomolgus CD8 ALG.APV-210 binding (nM) T cells T cells EC₅₀0.2 0.4 95% CI 0.17-0.21 0.37-0.44 HillSlope 2.3 3.4

The study showed that the EC₅₀ of the CD137-5T4 bispecific antibodyALG.APV-210 in a functional CD8 T cell activation assay was comparablebetween human (0.2 nM) and cynomolgus monkey (0.4 nM). The T cellactivation was mediated by crosslinking of CD137 which is dependent on5T4 in both human and cynomolgus CD8 T cell assays.

Example 16 In Vitro Activity of a 5T4-CD137 Bispecific AntibodyALG.APV-210 in a T-Cell IFN-γ Secretion and T Cell Proliferation Assays

The agonistic function of the 5T4-CD137 bispecific molecule ALG.APV-210at inducing target-dependent activation of CD137 was evaluated in aT-cell IFN-γ secretion assay and a T-cell proliferation assay usingunseparated peripheral blood mononuclear cells (PBMC). In order to testthe function of the anti-CD137 bispecific constructs, primary PMBC werestimulated with anti-CD3 antibodies to upregulate CD137, which is notexpressed, or is expressed at low levels, on resting T cells.Co-stimulation of primary T cells through the TCR and CD137 enhancesT-cell secretion of the cytokine interferon gamma (IFN-γ).

Methods

IFN-γ Secretion Assay:

Peripheral blood mononuclear cells (PBMC) were isolated from healthyhuman blood using standard ficoll gradients. The isolated cells werewashed in saline buffer to remove platelets. PBMC were cultured withCHO-K1 cells transfected with human 5T4 or empty vector, atapproximately 120,000 PBMC to 30,000 CHO-K1 cells in 96-well plates.Anti-CD3 antibody OKT3 (eBioscience) was added to all the wells at aconcentration of 1 ng/mL. Cells were cultured in a total volume of 200μL of RPMI 1640 media supplemented with 10% fetal bovine serum, sodiumpyruvate, antibiotics and non-essential amino acids. Plates wereincubated at 37° C., 5% CO₂ in humidified incubators for 72 hours. 100μL of media were collected after 72 hours of stimulation of PBMCcultures primed with a sub-optimal concentration of anti-CD3 antibodiesto induce upregulation of CD137. Levels of the IFN-γ were measured inthe supernatant using Milliplex® kits (Millipore) using themanufacturer's instructions. Data was collected in a Bio-Plex Reader 200System (Bio-Rad). Nonlinear regression analysis to determine EC₅₀ valueswas performed in GraphPad Prism 6® graphing and statistics software.

T-Cell Proliferation Assay:

PBMC were isolated from healthy human blood using standard ficollgradients. Once isolated, the cells were washed in saline buffer toremove platelets. PBMC were labeled with CellTrace™ Violet(Thermofisher), washed, and cultured with irradiated CHO-K1 cellstransfected with human 5T4 at approximately 120,000 PBMC to 30,000CHO-K1 cells in 96-well plates. CHO-K1 cells were irradiated (x-ray CellRad irradiator, Faxitron Bioptics, LLC), and washed in medium beforeplating with PBMC. Anti-CD3 antibody OKT3 (eBioscience) was added to allthe wells at a concentration of 5 ng/mL. Cells were cultured in a totalvolume of 200 μL of RPMI 1640 media (GIBCO) supplemented with 10% humanserum (SIGMA), sodium pyruvate, antibiotics and non-essential aminoacids. Plates were incubated at 37° C., 5% CO₂ in humidified incubatorsfor 96 hours.

Supernatants were removed and cells were labeled in the same assayplates with antibodies to CD4, CD8 and CD5 in PBS buffer with 2% BSA and2 mM EDTA, for 30 min on ice. 7AAD (SIGMA) was added to enable exclusionof dead cells in the analysis. After washing, cells were resuspended at120 μL/well, and 70 μL/well volumes were collected from each well andanalyzed in a flow cytometer (a LSR-II™, BD Biosciences). Data analysiswas performed using Flowjo software in two ways: calculating the % ofCD8+ live T cell events (CD4− CD8+ CD5+, 7AAD−) that had undergone atleast one cell division, or by counting the number of CD8+ live T cellevents (CD4− CD8+ CD5+, 7AAD−). In both cases the analysis was performedafter gating on lymphocytes using FSC versus SSC parameters. Eachcondition was tested in duplicates and the averages of each duplicateset were graphed using GraphPad Prism 6® graphing and statisticssoftware.

Results and Conclusions

Results of the IFN-γ secretion experiments are shown in FIG. 32. Everydata point in the graphs represents the average of duplicate wells.Addition of anti-CD3 alone, with no bispecific antibodies, induced verylow levels of IFN-γ. The addition of the ALG.APV-210 bispecific moleculeincreased the secretion of IFN-γ in a dose-dependent manner. Baselinelevels of IFN-γ induced by the anti-CD3 antibody, as well as the totalamount of IFN-γ secretion induced by ALG.APV-210 varied by PBMC donor(FIG. 32A v. FIG. 32B). Enhanced secretion of IFN-γ was observed in thepresence of CHO-K1 cells expressing 5T4, but not in the presence ofCHO-K1 cells transfected with the empty vector control. Therefore, thebispecific molecule ALG.APV-210 requires engagement of 5T4 to stimulateCD137 function as measured by IFN-γ secretion.

Results of the T cell proliferation experiments are shown in FIG. 33.Every data point in the graphs represents the average of duplicatewells. Polyclonal proliferation of CD8+ T cells was evaluated after 96hours of stimulation with priming at a sub-optimal concentration ofanti-CD3 antibody. Addition of anti-CD3 alone, with no bispecificantibody, induced low levels of CD8+ T-cell proliferation. The additionof ALG.APV-210 increased the proliferation of CD8+ T cells in adose-dependent manner (FIG. 33A and FIG. 33B). Both the percentages ofcells undergoing cell division (top panels) and the total cell counts(bottom panels) collected per well were consistent with robust celldivision and accumulation of divided CD8+ T cells in the wells. Theresults were consistent across multiple donors.

Example 17 Functional Reporter Activity of ALG.APV-210 in the Presenceof Various Human Tumor Lines Expressing a Range of 5T4 Protein Densities

Experiments were conducted to determine whether the ALG.APV-210construct has binding and functional reporter activity across multiplehuman tumor cell lines expressing 5T4 at a range of densities on thecell surface. The maximal binding capacity of ALG.APV-210 will varydepending on the cells' expression level of 5T4 tumor antigen. Bindingof ALG.APV-210 was therefore tested on 3 tumor cells lines (MDA-MB-231,H1975, and TF-1) naturally expressing a range of 5T4 densities, as wellas CHO-K1 cells stably transfected with 5T4 (CHO/5 T4).

Material and methods

5T4 binding assay: A 3-fold titration of ALG.APV-210 construct (rangingfrom 100 nM to 0.14 nM) was incubated with the human 5T4-expressing celllines for 40 minutes in FACS buffer at 4° C., washed 3 times, anddetected with PE-labeled, goat-anti-human Fc secondary antibody (JacksonImmunoResearch). Binding of ALG.APV-210 was detected by flow cytometry.All samples were run in duplicates. Nonlinear regression analysis todetermine EC₅₀ values was performed in GraphPad Prism 6® graphing andstatistics software.

5T4 protein density: The surface protein density of 5T4 present on eachcell line was examined using Quantibrite™ Beads (BD Pharmingen).Quantibrite™ Beads were acquired using the exact cytometer settings usedto acquire the cells for the binding curves with ALG.APV-210. The 4 beadpopulations were gated and the geometric mean fluorescence wasdetermined for each peak. A linear regression Log₁₀ of PE molecules perbead was plotted against the Log₁₀ fluorescence using the equationy=mx+c, where y equals Log₁₀ fluorescence and x equals Log₁₀ PEmolecules per bead. From that, the number of PE molecules (5T4) per cellcould be calculated. For this particular assay the equation wasy=1.036x−1.478. All fluorescence mean values were calculated using thegeometric mean of the samples, as suggested by the manufacturer'sprotocol.

CD137 reporter assay: Jurkat/CD137 transfectants carrying a luciferasereporter gene under the control of an NF-κB promoter (Promega) werecultured with H1975, TF-1, MDA-MB-231, or CHO/5T4 tumor cells at a ratioof 30,000 reporter cells to 60,000 target cells in 96-well plates.Five-fold concentrations of the ALG.APV-210 molecule, with finalconcentration ranging from 10 nM to 0.0006 nM, were added. Cells werecultured in a total volume of 100 μL of RPMI 1640 media supplementedwith 1% fetal bovine serum, sodium pyruvate, antibiotics andnon-essential amino acids. Plates were incubated at 37° C., 5% CO₂ inhumidified incubators for 5 hours. 100 μL of Bio-Glow buffer (Promega)was added to each well, mixed, and incubated for 10 minutes beforemeasuring luminescence. Luminescence was measured in a MicroBeta² 2450Microplate Counter (Perkin Elmer). All data points represent duplicatesamples. Nonlinear regression analysis to determine EC₅₀ values wasperformed in GraphPad Prism 6® graphing and statistics software. Everypoint in the curve represents the average of duplicate wells. The y-axisshows values in relative fluorescence units (RLU).

Results and Conclusions

Each of the tumor cell lines tested varied in the amount of 5T4 proteinbeing expressed on the cell surface as determined using Quantibrite™Beads. Thus, the ability of ALG.APV-210 to bind and be functionallyactive with a range of 5T4 expression could be examined.

ALG.APV-210 bound to a range of 5T4 cell surface-expressing tumor celllines. As shown in FIG. 34, CHO/5T4 have 10-fold higher 5T4 expressionthan the endogenously expressing 5T4 tumor cell lines. Of the threenon-transfected tumor cell lines, H1975 had the highest 5T4 expression,whereas TF-1 cells had the lowest 5T4 protein density. MDA-MB-231 cellsshowed intermediate 5T4 expression. Surface 5T4 protein densities werecalculated from the binding curves and show a range of molecules/cellfrom 17,000 to over 250,000. Table 27 shows 5T4 cell surface proteindensities and binding EC₅₀ values generated from the binding curves.Density of 5T4 expression on tumor cell lines was calculated using afluorescence standard.

TABLE 27 Summary of 5T4 densities and EC₅₀ ALG.APV-210 Tumor line 5T4molecules/cell EC₅₀ (nM) CHO/5T4 278000 10 H1975 43000 20 MDA.MB-23133000 21 TF-1 18000 71

FIG. 35 shows the results of the functional CD137 reporter assay. Asshown, ALG.APV-210 was sufficiently able to induce NF-κB signaling whencross-linking CD137 via binding to CHO/hu5T4, H1975, MDA.MB.231 or TF-1tumor lines, despite the range of cell surface 5T4 expression from17,000 to over 250,000. Surface 5T4 protein density had an impact onmaximum functional activity (Max RLU), with CHO/hu5T4 cells showing thehighest RLU values. However, the three tumor cell lines induced robustreporter function. The results indicate that ALG.APV-210 functions as anagonist in the presence of 5T4-positive cells expressing a range of 5T4densities. Table 28 shows the EC₅₀ and Max RLU induced with ALG.APV-210in the CD137 reporter assay in the presence of the various human5T4-expressing tumor lines.

TABLE 28 Summary of ALG.APV-210 EC₅₀ of and Max RLU Tumor line EC₅₀ (nM)Max RLU CHO/5T4 0.0092 3092200 H1975 0.037 2743132 MDA.MB-231 0.0542243297 TF-1 0.063 2751522

Example 18 In Vitro Activity of a 5T4-CD137 Bispecific Antibody in anIFNγ Release Assay Using Human CD8 T Cells and Human HCT116 Tumor Cells

The functional activity of the 5T4-CD137 bispecific antibody ALG.APV-210was evaluated in a CD8 T cell assay, where CD8+ T cells stimulated withαCD3 antibodies coated on beads were co-cultured in plates with humanHCT116 tumor cells expressing endogenous levels of 5T4.

Materials and Methods

Mitomycin C treatment of HCT116 cells: HCT116 tumor cells, a human coloncarcinoma cell line (ATCC® CCL-247™) expressing endogenous levels ofhuman 5T4 (receptor density=6.2×10⁴/cell, Table 21) were pre-treatedwith Mitomycin C (0.5 mg/mL, Sigma-Aldrich) at a concentration of 50μg/mL to hamper tumor cell overgrowth. After 45 min of incubation withMitomycin C in 37° C., cells were washed three times in R10 medium(RPMI, Gibco #61870010 containing 10% heat inactivated FBS, Hyclone#SV30160 lot RB35944 and 10 mM Hepes, Gibco #15630056) and thenresuspended at a concentration of 10×10⁶ cells/mL in new R10 medium. 50μL (5×10⁵ cells/well) of the Mitomycin C treated HCT116 cells were addedto TC treated assay plates (Eppendorf #0030 730.119) and incubated in37° C. overnight.

CD8 T cell assay setup: The following day, human PBMC were isolated bydensity gradient centrifugation using Ficoll-Paque (GE Healthcare#17-1440-02) from leucocyte concentrates obtained from healthy donors(Clinical Immunology and Transfusion Medicine, Labmedicin Region Skåne,Lund Sweden). CD8+ cells were enriched by negative selection using theCD8+ T cell isolation kit (Miltenyi 130-096-495). The bispecificantibody, ALG.APV-210, or an isotype control in the Adaptir format,ANC017 (variable heavy and light chain sequences for ANC017 are shownbelow in Tables 29A and 29B) was diluted in a serial dilution in R10medium and 50 μL was added to each well of the assay plates andincubated at 37° C. for 30 min, prior to the addition of 50 μL of αCD3beads (4×10⁸/mL of anti-CD3 antibodies clone: OKT-3, AffymetrixeBioscience #16-0037-85, coated on beads, diluted to 2×10⁶/mL) at a 1:1CD8+ T cell to beads ratio, followed by the addition of 50 μL of theeffector cells (enriched CD8+ cells, 2×10⁶/mL or 1×10⁵/well). The assayplates were incubated for 72 h at 37° C., and culture supernatantharvested. IFNγ levels in the supernatants were measured by ELISA (BDOptiEIA #555142) and are presented in FIG. 36 as normalized data (meanand SD values). EC₅₀ values were determined by non-linear regression log(agonist) vs. Normalized response (variable slope) using GraphPad Prism7. TOP value (maximal functional effect) was determined by calculatingthe mean of the two highest values of IFNγ production in the culturesupernatant. The experiment was performed 4 times, including in total 12donors.

TABLE 29A ACN017 Variable Region Sequences Heavy chain Light ChainAA sequence SEQ ID AA sequence SEQ ID CDR1 GFTFSSYA  30 QSISSY   8 CDR2ISGSGGST  32 AAS  10 CDR3 AKGSGSYFDL 177 QQYSGYPYT 178 VH/VLEVQLLESGGGLVQPGGSLRLSCAASGFT 179 DIQMTQSPSFLSASVGDRVTITCRA 180FSSYAMSWVRQAPGKGLEWVSAISGSGG SQSISSYLNWYQQKPGKAPKLLIYASTYYADSVKGRFTISRDNSKNTLYLQMN ASSLQSGVPSRFSGSGSGTDFTLTISLRAEDTAVYYCAKGSGSYFDLWGQGTL SSLQPEDFATYYCQQYSGYPYTFGQ VTVSS GTKLEIK

TABLE 29B Full-length ACN017 Sequence Full ACN017 Seq (VH-Fc-VL) SEQ IDEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV 181RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGSYFDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSGYPYTFGQGTKLEIKSSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSPSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGSYFDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSGYPYTFGQGTKLEIKR S

Results and Conclusions

The functional effect of the CD137-5T4 bispecific antibody ALG.APV-210was determined using human CD8+ T cells (the effector cells) co-culturedin plates with HCT116 cells. As shown in FIG. 36, the bispecificCD137-5T4 antibody ALG.APV-210 induced potent T cell activation,measured by IFNγ release, in a dose-dependent manner in the presence ofHCT116 cells expressing endogenous levels of 5T4. For determination ofEC₅₀, see Table 30, showing mean values and 95% CI of 12 donors from 4pooled experiments. The endogenous levels of 5T4 expressed by the HCT116cells is sufficient to induce a high agonistic functional effect ofALG.APV-210 as demonstrated in FIG. 37, where IFNγ levels of 6individual donors following treatment with ALG.APV-210 is compared withan isotype control (ANC017). The efficacy (maximal IFNγ response) isalso presented in FIG. 38 (fold change vs. isotype control, mean and SDof 12 donors).

TABLE 30 EC₅₀ of ALG.APV-210 in a CD8+ T cell assay in the presence ofHCT116 cells expressing human 5T4 Potency of ALG.APV-210 HCT116 T cellassay EC₅₀ (nM) 0.17 95% CI 0.14 to 0.21

Example 19 In Vivo Anti-Tumor Efficacy of a Bispecific Antibody inHCT-116 a 5T4 Positive Xenograft Tumor Model in SCID Beige Mice

A study was conducted to assess the anti-tumor efficacy in vivo ofALG.APV-210.

Materials and Methods

Female SCID beige mice (6-8 weeks old) from Taconic, Denmark were usedin the experiment. All animal procedures were conducted in compliancewith Swedish legislation on animal rights and protection, and approvedby the Ethical Committee on Animal Experiments, in Lund/Malmö (ethicalapplication number: M151-13). PBMC were isolated from leukocyteconcentrates obtained from 4 healthy donors (Clinical Immunology andTransfusion Medicine, Labmedicin Region Skåne, Lund Sweden) by densitygradient centrifugation using Ficoll-Paque (GE Healthcare #17-1440-02)according to the manufacturer's instructions. The HCT-116 cell line, ahuman colon carcinoma cell line positive for the tumor-associatedantigen 5T4, was purchased from ATCC (ATCC® CCL-247™, lot# 62765668) andcultured according to their recommendations.

On day 0, HCT-116 tumor cells, growing in log phase, were inoculatedsubcutaneously into the right hind flank of the mice (3×10⁶ in 110 μLPBS). The following day (day 1), 7×10⁶ of human PBMC from 4 donors in100 μL of PBS were injected intraperitoneally. Intraperitoneal antibodytreatments (100 μL in PBS) starting on day 6 with either vehicle (PBS),ALG.APV-210 10 μg/mouse, or ALG.APV-210 100 μg/mouse were giventwice/week for a total of 5 injections (n=5 mice/treatment/donor). Tumorgrowth was observed and measured twice/week with a caliper in width,length and height of which the tumor volume was calculated(w/2×1/2×h/2×pi×(4/3)). The experimental endpoint was tumor volume≥2cm³, wounding, or affected health of the mice. Tumor growth wasstatistically analyzed using Mann-Whitney non-parametric 2-tailed T-testusing GraphPad Prism 7 at different time points (day 9, 13, 16, 20 and23).

Results

As shown in FIG. 28, ALG.APV-210 at a dose of either 10 μg/mouse or 100μg/mouse showed statistically significant anti-tumor efficacy (e.g.tumor volume inhibition) in comparison with the vehicle group from day13 to 23 (pooled data from 4 donors, for statistical analysis andp-values; see Table 31).

TABLE 31 Statistical analysis of anti-tumor efficacy following treatmentwith ALG.APV-210 in an HCT-116 xenograft tumor model Days Significantdifference (p < 0.05) in tumor volume between following treatments,Mann-Whitney, non-parametric 2-tailed T test Tumor ALG.APV-210 10 μgALG.APV-210 100 μg inoculation vs. vehicle vs. vehicle Day 9 No No Day13 Yes * p = 0.0109 Yes * p = 0.0262 Day 16 Yes*** p = 0.0008 Yes** p =0.0010 Day 20 Yes**** p < 0.0001 Yes* p = 0.0009 Day 23 Yes** p = 0.0016Yes* p = 0.0225

In conclusion, the study showed that ALG.APV-210 had anti-tumoralproperties in a 5T4 positive human colon carcinoma xenograft tumormodel.

Example 20 Pharmacokinetic Properties of ALG.APV-209 in BALB/c Mice

A study was conducted to assess the pharmacokinetic (PK) properties ofALG.APV-209 and ALG.APV-210 in mice. Normal BALB/c female mice wereinjected intravenously (IV) at time 0 with a single dose of 200 μg ofALG.APV-209 or ALG.APV-210 and blood was collected from 1 to 3 mice pertime point. Anesthetized mice were exsanguinated via cardiac punctureand serum was collected at t=15 minutes, and 2, 6, 24, 48, 72, 96, 168,336 and 504 hours after injection. Concentrations of ALG.APV-209 andALG.APV-210 in sera were determined with an ELISA method developed todetect only intact protein, using huCD137 ECD-AFH (ALG027) to capturethe anti-CD137 BD, and biotinylated 5T4 ECD-mFc (ALG029) to detect theanti-5T4 BD. Estimated PK disposition parameters from non-compartmentalanalysis (NCA) using Phoenix 64 software (v6.4 WinNonlin™ license) arelisted in Table 32, and estimates of half-life along with fit statisticsare shown in FIG. 29. A precompiled model for IV dosing with sparsesampling and uniform weighting was used during NCA.

In normal female BALB/c mice injected IV with a 200 μg bolus dose ofALG.APV-209 or ALG.APV-210, the apparent terminal elimination half-lifedetermined using NCA was 142 and 215 hours, respectively (excludingsamples potentially impacted by ADA). Parameter estimates for half-lifedetermined using compartmental analysis (2-compartments with IV dosing)were similar for ALG.APV-209 and ALG.APV-210. NCA estimated clearanceand volume for ALG.APV-209 and ALG.APV-210 to be: 0.263 and 0.204mL/hr/kg, and 54 and 63 mL/kg respectively. By both analysis methods,ALG.APV-210 had the longest terminal elimination half-life and slightlybetter clearance values. Sudden drops in serum concentrations indicativeof anti-drug antibodies (ADA) were seen in late time points for micedosed with ALG.APV-210, however the presence of ADA could not beconfirmed by ELISA methods due to drug still being present in samples,which also likely resulted in rapid clearance of ADA.

TABLE 32 NCA estimated PK Disposition parameters of ALG.APV-209 andALG.APV-210 ALG.APV-210 PK Paramenter Units ALG.APV-209 ALG.APV-210excluding ADA + sera Rsq 0.82 0.900 0.966 HL Lambda z hr 142.0 134.6215.3 Tmax hr 0.25 0.25 0.25 Cmax μg/mL 254.04 252.34 252.34 SE Cmaxμg/mL N/A 14.6 14.6 Cmax/D kg*μg/mL/mg 0.025 0.025 0.025 C₀ μg/mL 262.2263.6 263.6 AUClast hr*μg/mL 34858 33054 38705 AUCINF_obs hr*μg/mL 3804336317 49124 AUCINF/D_obs hr*kg*μg/mL/mg 3.804 3.632 4.912 V-obs mL/kg53.84 53.46 63.22 CL_obs mL/hr/kg 0.263 0.275 0.204 Vss/OBS mL/kg 51.151.5 62.7 MRTlast hr 147.4 136.7 171.9 MRTINF-obs Hr 194.4 187.2 308.2Rsq: Goodness of fit statistic for the terminal elimination phase HLLambda z: Apparent terminal elimination half life Tmax: Time of maximumobserved concentration Cmax: Maximum observed concentration, occurringat Tmax SE Cmax: Standard error of the data at Tmax (time of maximummean concentration) Cmax/D: Maximum observed concentration divided bydose C₀: Back extrapolated initial concentration at time 0 _obs: basedon the observed concentrations AUClast: Area under the curve from thetime of dosing until the last measured concentration AUCINF: Area underthe curve from the time of dosing extrapolated to infinity AUCINF/D:AUCINF divided by dose V: Volume of distribution based on the terminalphase CL: Serum clearance Vss: An estimate of the volume of distributionat steady state MRTlast: Mean residence time until the last measuredconcentration MRTINF: Mean residence time extrapolated to infinity

Example 21 The Fc Portion of ALG.APV-210 Does Not Interact with FcγReceptors

ALG.APV-210 contains mutations introduced to prevent interaction withFcγ receptors (FcγR) that could lead to FcγR-mediated T-cell activationor ADCC, ADCP, CDC etc. Binding of ALG.APV-210 was therefore tested onCHO cell transfectants stably expressing the following FcγR: FcγRI(CD64), FcγRIIa (CD32a His131), FcγRIIb (CD32b), FcγRIIIa (CD16a Val158and CD16a Phe158), and FcγRIIIb (CD16b). Untransfected CHO cells wereused as negative control. A control human IgG1 molecule was used as apositive control. Different concentrations of ALG.APV-210 or controlIgG1 were incubated with the FcγR-expressing cell lines, washed, andlabeled with a secondary anti-human IgG reagent. Binding of ALG.APV-210and the control IgG1 was detected by flow cytometry.

The results are provided in FIGS. 30A and 30B. FIG. 30B shows that thecontrol IgG1 bound to all the tested FcγR expressing cell lines: FcγRI(CD64), FcγRIIa (CD32a His131), FcγRIIb (CD32b), FcγRIIIa (CD16a Val158and CD16a Phe158), and FcγRIIIb (CD16b), but not to the untransfectedCHO cells (negative control). Significant binding of the control IgG1 tosome of the FcγR expressing cell lines were observed at concentrationsas low as 4 nM. FIG. 30A shows that no detectable binding was observedwith the ALG.APV-210 construct to the tested FcγR: FcγRI (CD64), FcγRIIa(CD32a His131), FcγRIIb (CD32b), FcγRIIIa (CD16a Val158 and CD16aPhe158), and FcγRIIIb (CD16b).

Example 22 In Vitro Activity of a 5T4-CD137 Bispecific Antibody in aT-Cell Proliferation Assay Using Human PBMC and CHO-5T4 Cells

The functional activity of the 5T4-CD137 bispecific moleculesALG.APV-209 and ALG.APV-210 was evaluated in a T-cell proliferationassay using PBMC. PBMC were isolated from healthy human blood usingstandard ficoll gradients. Once isolated, the cells were washed insaline buffer to remove platelets. PBMC were cultured with irradiatedCHO-K1/human 5T4 cells, at approximately 120,000 PBMC to 30,000 5T4 (+)cells in 96-well plates. CHO-K1/5T4 cells were irradiated (x-ray CellRad irradiator, Faxitron Bioptics, LLC), and washed in medium beforeplating with PBMC. Anti-CD3 antibody OKT3 (eBioscience) was added to allthe wells at a concentration of 1 ng/mL. Cells were cultured in a totalvolume of 200 μL of RPMI 1640 media (GIBCO) supplemented with 10% fetalbovine serum (SIGMA), sodium pyruvate, antibiotics and non-essentialamino acids. Plates were incubated at 37° C., 5% CO2 in humidifiedincubators for 72 hours. Supernatants were removed and cells werelabeled in the same assay plates with antibodies to CD4, CD8 and CD5 inPBS buffer with 2% BSA and 2 mM EDTA, for 30 min on ice, 7AAD was addedto exclude dead cells. After washing cells were resuspended at 120μL/well and 70 μL/well volumes were collected from each well in a flowcytometer (a LSR-II™, BD Biosciences). Cell analysis was performed usingFlowjo software and analyzed by counting the number of CD4+ or CD8+ liveT cell events (CD4+ CD8− CD5+, 7AAD− or CD4− CD8+ CD5+ 7AAD−) eventsafter gating on lymphocytes using FSC versus SSC parameters. Eachcondition was tested in duplicate and the averages of each duplicate setwere graphed using GraphPad Prism 6® graphing and statistics software.

The results of the study are provided in FIGS. 31A and 31B.Antigen-specific proliferation of CD8+ T cells was evaluated after 72hrs of stimulation of T cells primed with a low concentration ofanti-CD3 antibodies. Anti-CD3 stimulation induces upregulation of CD137.Addition of anti-CD3 alone, with no bispecific antibodies, induced acertain amount of CD8+ and CD4+ T-cell proliferation. The addition ofALG.APV-209 or ALG.APV-210 increased the proliferation of CD8+ T cells(FIG. 31A). In contrast, and as expected based on the preferentialexpression of CD137 on CD8+ T cells, the bispecific antibodies had alimited impact on the anti-CD3 induced proliferation of CD4+T cells(FIG. 31B). The results were consistent across multiple experiments. AMorrison format control molecule (ALG.APV-004) was included forcomparison.

Taken together, the results showed that both ALG.APV-209 and ALG.APV-210enhanced the proliferation of CD8+ T cells. ALG.APV-209 and ALG.APV-210induced a higher amount of cell proliferation than the Morrison formatconstruct ALG.APV-004.

Example 23 5T4 Expression in Normal and Tumor Tissues Assessed byImmunohistochemistry

Expression of the tumor antigen 5T4 in normal human tissue and in arange of different tumor types was assessed by immunohistochemistry(IHC).

Development of staining method: Five commercial anti-5T4 antibodies andtwo in-house generated anti-5T4 antibodies were assessed for 5T4-bindingin cryosections versus formalin-fixed, paraffin-embedded (FFPE) sectionsof the control cells and test human tissues. Isotype control stainingwas also included. A mouse monoclonal antibody (clone: MAB4975, R&DSystems) was selected as the best performing antibody because it washighly specific and functional in both cryosections and in FFPEsections. Antigen retrieval method and antibody titer were optimized forthis antibody.

Tissues and tissue microarrays: Human placental tissue and a murinecolon carcinoma cell line CT26 (from ATCC® CRL-2638) transfected toexpress human 5T4 were used as positive controls. Human cerebrum, liver,and non-transfected CT26 cells were used as negative controls. Humantissues used for positive and negative controls were from Charles RiverLaboratories' archives. FFPE tissue microarrays (TMAs) were acquiredfrom US Biomax Inc. All stainings were evaluated by a study pathologistat PAI, Charles River Laboratories.

IHC staining of normal human tissue microarrays: In the normal humantissue panel with 24 tissue types from 3 donors (FDA808j-1), 5T4expression was not found in any major organs.

IHC staining in human tumor tissue types: 5T4 expression was screened inthe multiple organ cancer tissue array of 72 tumor cases (FDA808j -2)presented by a single core per tumor. 5T4 expression was detected intumors of the pancreas, thyroid gland, breast, liver, uterus, cervix,striated muscle, skin (squamous cell carcinoma), nerve (neurofibroma),and bladder.

5T4 expression was assessed in TMAs containing 10-50 tumor cases foreach of the selected indications shown in Table 33, represented byduplicate or triplicate cores. In bladder cancer, cancer of the head andneck (H&N), non-small cell lung cancer (NSCLC) and mesothelioma, 50-56%of the tumors investigated were 5T4 positive. A significant number ofpancreatic tumors were also 5T4 positive (36%). 50% of the clear cellcarcinomas tumors investigated were 5T4 positive.

TABLE 33 5T4 expression in seven selected tumor indications 5T4 + 5T4 +Total No. donors donors TMA ID donors (Total #) (%) Bladder cancer BL72118 10 56 Head & neck cancers HN811 19 10 53 Non-small cell lung LC10011a40 20 50 cancer Mesothelioma MS481c 20 10 50 Pancreatic cancer PA721a 228 36 Kidney cancer T072b 10 2 20 Ovarian cancer T112c 10 1 10

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not, be taken as an acknowledgment orany form of suggestion that they constitute valid prior art or form partof the common general knowledge in any country in the world.

1.-95. (canceled)
 96. A multispecific polypeptide comprising a firstscFv domain and a second scFv domain, wherein the first and second scFvdomains are linked together by a binding domain linker or a bindingdomain linker and an immunoglobulin Fc domain, wherein theimmunoglobulin Fc domain comprises a hinge region and an immunoglobulinconstant region; and wherein the second scFv domain specifically bindsto human 4-1BB and comprises: (i) an immunoglobulin heavy chain variableregion comprising an HCDR1 amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 18, 24, an HCDR2 amino acid sequence of SEQID NO: 4, and an HCDR3 amino acid sequence of SEQ ID NO: 6; and (ii) animmunoglobulin light chain variable region comprising an LCDR1 aminoacid sequence of SEQ ID NO: 8, an LCDR2 amino acid sequence of SEQ IDNO: 10, and an LCDR3 amino acid sequence of SEQ ID NO:
 12. 97.-99.(canceled)
 100. The multispecific polypeptide of claim 96, wherein themultispecific polypeptide comprises, from amino-terminus tocarboxyl-terminus, (i) the first scFv domain, (ii) a binding domainlinker, and (iii) the second scFv domain.
 101. The multispecificpolypeptide of claim 96, wherein the multispecific polypeptidecomprises, from amino-terminus to carboxyl-terminus, (i) the second scFvdomain, (ii) a binding domain linker, and (iii) the first scFv domain.102. The multispecific polypeptide of claim 96, wherein themultispecific polypeptide comprises, from amino-terminus tocarboxyl-terminus, (i) the first scFv domain, (ii) a hinge region, (iii)an immunoglobulin constant region, (iv) a binding domain linker, and (v)the second scFv domain. 103.-104. (canceled)
 105. The multispecificpolypeptide of claim 96, wherein the binding domain linker comprises anamino acid sequence selected from SEQ ID NOs: 85-108. 106.-109.(canceled)
 110. The multispecific polypeptide of claim 96, wherein thesecond scFv domain comprises a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 14,20, 26, and 28 and a light chain variable region selected from the groupconsisting of SEQ ID NO: 16 and
 22. 111.-113. (canceled)
 114. Themultispecific polypeptide of claim 96, wherein the first scFv domaincomprises (i) an immunoglobulin heavy chain variable region comprisingan HCDR1 amino acid sequence selected from the group consisting of SEQID NOs: 30, 52, and 60, an HCDR2 amino acid sequence selected from thegroup consisting of SEQ ID NOs: 32 and 62, and an HCDR3 amino acidsequence of SEQ ID NO: 34; and (ii) an immunoglobulin light chainvariable region comprising an LCDR1 amino acid sequence selected fromthe group consisting of SEQ ID NOs: 8, 42, and 54, an LCDR2 amino acidsequence of SEQ ID NOs: 10, and an LCDR3 amino acid sequence of SEQ IDNOs: 36; and wherein the second scFv domain comprises (i) animmunoglobulin heavy chain variable region comprising an HCDR1 aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 18,24, an HCDR2 amino acid sequence selected of SEQ ID NO: 4, and an HCDR3amino acid sequence of SEQ ID NO: 6; and (ii) an immunoglobulin lightchain variable region comprising an LCDR1 amino acid sequence of SEQ IDNO: 8, an LCDR2 amino acid sequence of SEQ ID NO: 10, and an LCDR3 aminoacid sequence of SEQ ID NO:
 12. 115.-126. (canceled)
 127. Themultispecific polypeptide of claim 96, wherein the first scFv domaincomprises i) a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 38, 46, 56,and 64 and a light chain variable region comprising an amino acidselected from the group consisting of SEQ ID NO: 40, 44, 48, 50, 58, 68,and 70, and wherein the second scFv domain comprises ii) a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14, 20, 26, and 28, and a light chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 16 and
 22. 128.-135. (canceled)
 136. Themultispecific polypeptide of claim 96, wherein the first scFv domainspecifically binds to human 5T4 and comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 118, 120, 122, 124,126, 128, 130, 132, 134, and 170; and wherein the second scFv domainspecifically binds to human 4-1BB and comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 110, 112, 114, and 116.137.-147. (canceled)
 148. The multispecific polypeptide of claim 96,wherein the polypeptide comprises an amino acid sequence comprising atleast 80%, at least 85%, at least 90%, or at least 95% sequence identityto a sequence selected from the group consisting of SEQ ID NOs: 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 172, and 174.149.-150. (canceled)
 151. The multispecific polypeptide of claim 96,wherein the first scFv domain binds to an extracellular domain of 5T4and wherein the second scFv domain binds to an extracellular domain of4-1BB. 152.-174. (canceled)
 175. The multispecific polypeptide of claim96, wherein binding of the multispecific polypeptide to an effector cellresults in enhanced effector cell activation. 176.-177. (canceled) 178.The multispecific polypeptide of claim 96, wherein the 5T4-bindingdomain is capable of binding 5T4 with a kD value of less than 50 nM.179. The multispecific polypeptide of claim 96, wherein the light chainvariable region and/or the heavy chain variable region of the5T4-binding domain or the 4-1BB-binding domain is humanized. 180.-185.(canceled)
 186. A method for treating a cancer in a subject, whereinsaid cancer is characterized by expression of 5T4, the method comprisingadministering to the subject a therapeutically effective amount of themultispecific polypeptide of any one of claim
 96. 187. (canceled) 188.The method of claim 186, wherein the cancer is breast cancer, pancreaticcancer, ovarian cancer, non-small cell lung cancer, mesothelioma,chronic lymphocytic leukemia (CLL), mantle cell leukemia (MCL), acutelymphoblastic leukemia (ALL), squamous cell carcinoma, melanoma, adrenalcancer, bladder cancer, cervical cancer, renal cancer, gastric cancer,prostate cancer, thyroid cancer, liver cancer, uterine cancer,neurofibroma, sarcoma, carcinoma, or head and neck cancer. 189.-200.(canceled)